WO2017130630A1 - Agent d'amélioration de la résistance d'une plante aux températures élevées, procédé d'amélioration de la résistance aux températures élevées, agent de suppression de blanchiment, et un promoteur d'expression de gène dreb2a - Google Patents

Agent d'amélioration de la résistance d'une plante aux températures élevées, procédé d'amélioration de la résistance aux températures élevées, agent de suppression de blanchiment, et un promoteur d'expression de gène dreb2a Download PDF

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WO2017130630A1
WO2017130630A1 PCT/JP2016/089061 JP2016089061W WO2017130630A1 WO 2017130630 A1 WO2017130630 A1 WO 2017130630A1 JP 2016089061 W JP2016089061 W JP 2016089061W WO 2017130630 A1 WO2017130630 A1 WO 2017130630A1
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Prior art keywords
plant
temperature stress
allantoin
plants
gene
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PCT/JP2016/089061
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English (en)
Japanese (ja)
Inventor
坂本　敦
裕士 島田
翔真 田中
増俊 野尻
ユウ 付
憲之 木崎
北野　光昭
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株式会社カネカ
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Priority claimed from JP2016043503A external-priority patent/JP6532026B2/ja
Application filed by 株式会社カネカ filed Critical 株式会社カネカ
Priority to CN201680080335.6A priority Critical patent/CN108882712B/zh
Priority to EP16888214.0A priority patent/EP3409116B1/fr
Priority to ES16888214T priority patent/ES2955007T3/es
Publication of WO2017130630A1 publication Critical patent/WO2017130630A1/fr
Priority to US15/977,890 priority patent/US10182569B2/en
Priority to US16/034,010 priority patent/US20180310565A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/28—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N<
    • A01N47/36—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N< containing the group >N—CO—N< directly attached to at least one heterocyclic ring; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/28—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N<
    • A01N47/38—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N< containing the group >N—CO—N< where at least one nitrogen atom is part of a heterocyclic ring; Thio analogues thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Definitions

  • the present invention relates to a high-temperature stress tolerance improver in plants, a method for improving high-temperature stress tolerance, a whitening inhibitor, and a DREB2A gene expression promoter.
  • Plants are exposed to various environmental stresses such as high temperature and dryness. Since plants are difficult to move like animals, mechanisms have been developed to protect themselves from environmental stress.
  • a drought stress responsive element is a sequence whose presence was confirmed in the Arabidopsis genome by promoter analysis of RD29A, which is one of water stress-inducible genes.
  • DREB DRE binding protein
  • DREB2A is a transcription factor isolated as a protein that binds to DRE.
  • DREB2A is a transcription factor of APETALA2 / ethylene-responsive element binding factor type (AP2 / ERF-type) and was isolated as a protein that recognizes DRE (Non-patent Document 3). Since DREB2A is strongly induced by dry and high salt concentration conditions, it is considered that DREB2A is a transcription factor that works under dry and high salt stress conditions (Non-Patent Document 1).
  • DREB2A The activity of DREB2A is controlled after translation, and the 30 amino acid region adjacent to the AP2 / ERF DNA binding domain is thought to play an important role in the post-translational control of the protein.
  • DREB2A CA lacking this region has activity, and Arabidopsis overexpressing DREB2A CA exhibits a fertile phenotype, and drought stress tolerance has been significantly improved (non-patent literature). 2).
  • Non-Patent Document 3 and Patent Document 1 report a mechanism by which DREB2A induces a target gene when a plant is subjected to high temperature stress.
  • a mechanism has been proposed in which the protein DPB3-1 (or NF-YC10) interacts with DREB2A to promote induction of a high-temperature stress resistance gene by DREB2A. Yes.
  • Non-Patent Document 3 shows that the interaction between DPB3-1 and DREB2A does not affect the expression of a drought stress-inducing gene.
  • Patent Document 2 describes that in a transformed plant into which the DREB2A gene has been introduced, the rooting rate is improved and the longevity of cut flowers is prolonged.
  • allantoin (5-ureidohydantoin) is an intermediate product generated in the process of decomposing nucleobase (purine base).
  • allantoin is produced from 5-hydroxyisouric acid by allantoin synthase (AS) and decomposed into allantoic acid by allantoinase (ALN).
  • AS allantoin synthase
  • ALN allantoinase
  • Non-Patent Document 5 reports that growth is promoted when allantoin is given to Arabidopsis thaliana.
  • Patent Document 3 discloses a method for protecting a plant from stress by giving a ureido such as allantoin to the plant.
  • Patent Document 3 discloses that plants are damaged by oxidative stress during environmental disturbances such as drought and cold, and that when the concentration of ureido is high, the scavenger pathway is promoted and the plants are protected from damage.
  • allantoin is known to have an effect of improving stress tolerance such as drought stress tolerance and low temperature stress tolerance in plants.
  • stress tolerance such as drought stress tolerance and low temperature stress tolerance in plants.
  • high temperature stress tolerance and allantoin has not been investigated so far.
  • Non-Patent Document 3 As described above, interaction between DPB3-1 and DREB2A is necessary for induction of high-temperature stress tolerance by DREB2A, whereas this interaction is necessary for induction of drought stress tolerance. It is described as irrelevant. As is clear from this, even if a certain component can improve resistance to stress other than high temperature stress such as drought stress, it does not necessarily have an effect of improving high temperature stress resistance. Therefore, from the conventional knowledge that allantoin has an effect of improving tolerance to several stresses other than high-temperature stress in plants, the action of allantoin related to high-temperature stress tolerance cannot be predicted.
  • the expression of the DREB2A gene in plants contributes to improvement of various stress tolerances such as high temperature stress tolerance and drought stress tolerance as described above. It is also known that the expression of the DREB2A gene has advantageous effects such as an improved rooting rate and an extended flower life of cut flowers. A substance capable of promoting the expression of the DREB2A gene can exert these advantageous effects when applied to plants. However, no such material has been previously provided. As described in Patent Document 1, a transformed plant into which the DREB2A gene has been introduced can exert advantageous effects due to DREB2A, but because the transformed plant is a plant that does not exist in nature, it is difficult to use industrially. There's a problem. It is considered that the utility of a technique for improving the expression of the DREB2A gene in a plant by applying a naturally occurring substance to the plant is considered to be great.
  • an object of the present invention is to provide means for improving tolerance to high temperature stress in plants. Another object of the present invention is to provide a means for suppressing plant whitening. Another object of the present invention is to provide a means for promoting the expression of the DREB2A gene in plants.
  • the present inventors have found that when allantoin is allowed to act on a plant, an unexpected effect of improving the high temperature stress tolerance of the plant, suppressing the whitening of the plant, and promoting the DREB2A gene in the plant occurs.
  • the invention has been completed. Specifically, the present invention includes the following inventions.
  • the high temperature stress according to (1) for suppressing one or more of whitening of the plant due to high temperature stress, wiping of the plant due to high temperature stress, and winding of a plant leaf due to high temperature stress. Tolerance improver.
  • the high-temperature stress tolerance improving agent according to (1) or (2) which promotes the expression of the DREB2A gene in a plant.
  • a method for improving the high temperature stress tolerance of a plant comprising a step of applying the high temperature stress tolerance improving agent according to any one of (1) to (3) to the plant.
  • the method according to (4) wherein in the step, the high-temperature stress tolerance improver is applied to a plant before being subjected to stress.
  • a DREB2A gene expression promoter for promoting the expression of the DREB2A gene in a plant, containing allantoin as an active ingredient.
  • a method for suppressing whitening of a plant comprising a step of applying the whitening inhibitor according to (6) to a plant.
  • a method for promoting the expression of the DREB2A gene in a plant comprising a step of applying the DREB2A gene expression promoting agent according to (7) to the plant.
  • Use of allantoin to improve high temperature stress tolerance of plants (11) Use of allantoin to suppress plant whitening.
  • FIG. 1 photographs of each petri dish after Arabidopsis thaliana was treated under each heat shock condition and grown for 1 week are shown.
  • the survival rate when Arabidopsis thaliana is treated under each heat shock condition and grown for 1 week is shown.
  • Experiment 2 the photograph of the onion of the 3rd day after the heat shock processed for 1.5 hours at 45 degreeC is shown.
  • the upper part of FIG. 3 is a photograph of an onion in the allantoin area (survival rate 33.3%), and the lower part of FIG. 3 is a photograph of the onion in the water area (survival rate 0%).
  • Allantoin is also called 5-ureidohydantoin, and has a structure in which a free form is represented by the following formula.
  • Allantoin has one asymmetric carbon (indicated by * in the formula), and there are (R) -allantoin and (S) -allantoin forms.
  • the allantoin used in the present invention may be (R) -allantoin, (S) -allantoin, or a mixture thereof.
  • Allantoin may be in any form that can be used by plants, and can be in an appropriate form such as a free form or a solvate such as a hydrate. Moreover, 2 or more types of mixtures may be sufficient among forms, such as a free body and a hydrate.
  • the target plant to which the allantoin, the allantoin-containing composition, the high-temperature stress tolerance improver, the bleaching inhibitor or the DREB2A gene expression promoter is applied is not particularly limited and includes various plants such as dicotyledonous plants and monocotyledonous plants.
  • dicotyledonous plants examples include morning glory plants, Brassica plants, convolvulaceae plants, sweet potato plants, Arabidopsis plants, papaver plants, pteridophytes plants, chickweed plants, papaver plants, prunus plants, clover plants , Nominotsunuri genus plant, Oyafusuma genus plant, Waspionian genus plant, Hamachabe genus plant, Otsumexa genus plant, Shiotsume genus plant, Mantema genus plant, genus genus plant, Fusiflora genus plant, Nadesicoaceae plant, euphorbiaceae plant, Asteraceae plant, Pepperaceae plant, Cypridaceae plant, Willow family plant, Prunus plant plant, Walnut plant plant, Birch plant plant, Beech plant plant, Elmaceae plant, Mulberry plant plant, Nettle plant plant plant, Chrysanthemum plant plant, Papaver plant Plant, sandal
  • Monocotyledonous plants include, for example, duckweed plants, duckweed plants, cattleya plants, cymbidium plants, dendrobum plants, leek plants, phalaenopsis plants, banda plants, paphiopedilum plants, orchidaceae plants, gammaidae Plants, licorices, periwinkles, cicadaceae, euphorbiaceae, euphorbiaceae, stigmaceae, scorpionaceae, cyperaceae, palmaceae, taroaceae, asteraceae, cypressaceae, It is applied to scorpionaceae plants, rush family plants, sandalaceae plants, liliaceae plants, amaryllidaceae plants, genus genus plants, iris plants, scallops plants, ginger plants, cannaid plants, cynomolgus plants, etc. Can do.
  • the target plant is not limited to a wild type plant, and may be a mutant or a transformant.
  • High temperature stress tolerance improving agent, method for improving high temperature stress tolerance> is a high temperature stress tolerance improver for improving the high temperature stress tolerance of a plant, containing allantoin as an active ingredient.
  • One embodiment of the present invention is a method for improving the high temperature stress tolerance of a plant, comprising the step of applying the above-mentioned high temperature stress tolerance improving agent to a plant.
  • the high temperature stress is stress caused by exposure of a plant to a temperature higher than the normal growth temperature, for example, 25 ° C. or higher, more specifically 30 ° C. or higher, and more specifically 50 This is stress caused by exposure of plants to temperatures below °C.
  • a temperature higher than the normal growth temperature for example, 25 ° C. or higher, more specifically 30 ° C. or higher, and more specifically 50
  • This is stress caused by exposure of plants to temperatures below °C.
  • it does not specifically limit as the time per day when a plant body is exposed to the said high temperature, For example, 60 minutes or more, More specifically, 90 minutes or more are mentioned, More specifically, 600 minutes or less is mentioned.
  • the high-temperature stress tolerance improving agent and the high-temperature stress tolerance improving method of the present invention are effective for improving tolerance to stress received by plants exposed to a high-temperature environment, particularly in a high-temperature stress. It is.
  • the high temperature stress tolerance improving agent and the high temperature stress tolerance improving method of the present invention are characterized in that, among the above physiological disorders caused by high temperature stress, at least the plant is whitened by high temperature stress (chlorosis), and high temperature stress. It has been confirmed that one or more of the plant withering and suppression of plant leaf rolling due to high temperature stress were suppressed.
  • the mechanism of the high-temperature stress tolerance enhancement action by allantoin is not particularly limited, but as one possibility, high temperature is promoted by promoting the expression of the DREB2A gene in plants.
  • the mechanism that stress tolerance improves is estimated.
  • the promotion of the expression of the DREB2A gene the following ⁇ 5.
  • the expression of the DREB2A gene is promoted in plants, and the heat shock transcription factor 3 ( It is estimated that the high temperature stress tolerance is improved by suppressing the expression of the HSF3) gene.
  • Non-Patent Document 1 in Arabidopsis thaliana in which DREB2A CA is overexpressed, the expression level of the HSF3 gene is improved, and accordingly, high-temperature stress tolerance is improved.
  • the expression level of the DREB2A gene is improved, while the expression level of the HSF3 gene is suppressed and high-temperature stress tolerance is improved, so that allantoin has been known so far. It is presumed that high temperature stress tolerance in plants is improved by a mechanism different from the mechanism.
  • the suppression of the expression of the HSF3 gene the following ⁇ 5.
  • the high-temperature stress tolerance improver of the present invention may be any agent that contains allantoin and has an effect of improving the high-temperature stress tolerance of plants, and may be allantoin itself or a combination of allantoin and other components.
  • the allantoin-containing composition may be used.
  • the high temperature stress tolerance improving agent of the present invention can contain an effective amount of allantoin that improves high temperature stress tolerance in plants.
  • the high-temperature stress resistance improver of the present invention can be in any shape such as solid or liquid.
  • the high-temperature stress tolerance improver of the present invention is an allantoin-containing composition
  • it may further contain other components useful for plants and components necessary for formulation.
  • other components include known fertilizer components.
  • Components necessary for formulation include carriers, liquid media and the like.
  • the high-temperature stress resistance improver of the present invention is an allantoin-containing composition
  • its production method is not particularly limited, and each component is mixed, or if it is a solid composition, it is pulverized, granulated, and dried as necessary. If it is a liquid composition, operations such as stirring and dispersion can be performed as necessary.
  • the method for improving the high temperature stress resistance of a plant according to the present invention includes a step of applying the above high temperature stress resistance improving agent to a plant.
  • the target plant is a plant that needs to be improved in resistance to high temperature stress, for example, a plant cultivated in an environment that may be subjected to high temperature stress such as the above temperature condition.
  • Specific plant types are as described above.
  • the high temperature stress tolerance improver of the present invention As a method of applying the high temperature stress tolerance improver of the present invention to plants, plants such as plant roots, stems, leaves, etc. in which the allantoin released from the high temperature stress tolerance improver of the present invention or the high temperature stress tolerance improver of the present invention is used.
  • the method is not particularly limited as long as it can contact the body, and may be applied so that the high-temperature stress tolerance improver of the present invention is in direct contact with the plant body, or cultivation of soil or the like on which the plant body is established You may apply the high temperature stress tolerance improvement agent of this invention to a support
  • the high-temperature stress tolerance improver of the present invention can be applied to plants so that an effective amount of allantoin that improves high-temperature stress tolerance is applied in plants.
  • the timing for applying the high-temperature stress tolerance improver of the present invention to plants is not particularly limited, but preferably, the high-temperature stress tolerance improver of the present invention is applied to plants before being subjected to high-temperature stress.
  • the high temperature stress tolerance improving agent of the present invention promotes the expression of the DREB2A gene. As described above, when the DREB2A gene is expressed, it is presumed that the expression of various stress resistance genes is induced. For this reason, it is estimated that the plant to which the high temperature stress tolerance improving agent of the present invention has been applied in advance before being subjected to high temperature stress is in a state in which high temperature stress tolerance has been improved in advance, and the survival rate when subsequently subjected to high temperature stress is high.
  • the time when the high temperature stress tolerance improver of the present invention is applied to the plant is T1, and the plant begins to be exposed to high temperature stress.
  • T2 the time from T1 to T2 is preferably 0.5 to 10 days, preferably 0.5 to 9 days, preferably 0.5 to 8 days, preferably 0.5 to 7 days, preferably Is 0.5-6 days, preferably 0.5-5 days, preferably 0.5-4 days, preferably 0.5-3 days, preferably 0.5-2 days, preferably 0.5-days. 1.5 days.
  • the survival rate after exposure to high temperature stress is high.
  • the high temperature stress tolerance improver of the present invention may be applied N times (N is 2 or more) to a plant before being subjected to high temperature stress. In that case, each time point T1 n (n is It is preferable that the time from an integer of 1 to N) to T2 satisfy the above range.
  • One embodiment of the present invention is a whitening inhibitor for suppressing plant whitening, which contains allantoin as an active ingredient.
  • One embodiment of the present invention is a method for suppressing plant whitening, which comprises the step of applying the whitening inhibitor to a plant.
  • Plant whitening also called chlorosis, is mainly caused by high-temperature stress.
  • the plant that is subject to whitening suppression according to the present invention is a plant that requires whitening suppression, for example, a plant that is cultivated in an environment that may be subjected to high-temperature stress such as the above temperature conditions.
  • Specific plant types are as described above.
  • the whitening inhibitor of the present invention only needs to contain allantoin and has an action of suppressing plant whitening, may be allantoin itself, or may contain allantoin and other components. It may be a composition.
  • the whitening inhibitor of the present invention can contain an effective amount of allantoin that suppresses whitening in plants.
  • the whitening inhibitor of the present invention can be in any shape such as solid or liquid.
  • the whitening inhibitor of the present invention is an allantoin-containing composition
  • the same allantoin-containing composition as described for the high-temperature stress resistance improver can be used.
  • the whitening inhibitor of the present invention or the allantoin released from the whitening inhibitor of the present invention may come into contact with a plant body such as a plant root, stem or leaf.
  • the method is not particularly limited as long as it can be applied, and may be applied so that the whitening inhibitor of the present invention is in direct contact with the plant body, or the whitening inhibitor of the present invention is applied to a cultivation carrier such as soil in which the plant body is fixed. May be applied.
  • the whitening inhibitor of this invention can be applied to a plant so that the effective amount of allantoin which suppresses whitening may be applied in a plant.
  • the timing for applying the whitening inhibitor of the present invention to plants is not particularly limited, but preferably, the whitening inhibitor of the present invention is applied to plants before being subjected to stress that causes whitening (for example, high-temperature stress).
  • the whitening inhibitor of the present invention promotes the expression of the DREB2A gene. As described above, when the DREB2A gene is expressed, it is presumed that the expression of various stress resistance genes is induced. For this reason, the plant to which the whitening inhibitor of the present invention has been applied in advance before being subjected to stress is in a state in which stress tolerance has been improved in advance, and it is predicted that whitening when subsequently subjected to stress is effectively suppressed. .
  • the time when the whitening inhibitor of the present invention is applied to the plant is T1, and the plant begins to be exposed to the stress.
  • the time from T1 to T2 is preferably 0.5 to 10 days, preferably 0.5 to 9 days, preferably 0.5 to 8 days, preferably 0.5 to 7 days, Preferably 0.5 to 6 days, preferably 0.5 to 5 days, preferably 0.5 to 4 days, preferably 0.5 to 3 days, preferably 0.5 to 2 days, preferably 0.5 ⁇ 1.5 days.
  • the whitening inhibitor of the present invention may be applied N times (N is 2 or more) to the plant before receiving the stress.
  • N is 2 or more
  • One embodiment of the present invention is a DREB2A gene expression promoter for promoting the expression of the DREB2A gene in plants, containing allantoin as an active ingredient.
  • One embodiment of the present invention is also a method of promoting the expression of the DREB2A gene in a plant, comprising the step of applying the above-mentioned DREB2A gene expression promoter to the plant.
  • inventions of the present invention contribute to the improvement of various stress tolerances such as high temperature stress tolerance and drought stress tolerance by promoting the expression of the DREB2A gene in plants. Moreover, there are advantageous effects such as improvement of the rooting rate in the target plant and extension of the flower life of cut flowers.
  • the expression of the HSF3 gene is preferably suppressed.
  • gene expression includes a process of expressing mRNA using genomic DNA as a template (transcription) and / or a process of synthesizing protein using the mRNA as a template (translation).
  • a gene is a nucleic acid containing a base sequence encoding a predetermined polypeptide, and typically refers to genomic DNA of a plant or mRNA generated using genomic DNA as a template.
  • Acceleration of the expression of the DREB2A gene in the present invention means that the expression level of the DREB2A gene is significantly increased as compared with a plant not applied with allantoin.
  • the promotion of the expression of the DREB2A gene in a plant can be confirmed by detecting the increase in the amount of mRNA encoding the DREB2A polypeptide and / or the amount of the DREB2A polypeptide in the plant. .
  • the degree of promotion of the expression of the DREB2A gene in the present invention is not particularly limited, but when the expression level of the DREB2A gene in a plant not applied with the DREB2A gene expression promoter of the present invention is 100, the expression of the DREB2A gene of the present invention
  • the expression level of the DREB2A gene in the plant to which the promoter is applied is, for example, 120 or more, preferably 150 or more, preferably 200 or more.
  • ⁇ Suppression of HSF3 gene expression in the present invention means that the expression level of the HSF3 gene is significantly reduced as compared with plants not applied with allantoin. Suppression of HSF3 gene expression in plants can be confirmed by detecting a decrease in the amount of mRNA encoding the HSF3 polypeptide and / or a decrease in the amount of HSF3 polypeptide in the plant. .
  • the degree of suppression of the expression of the HSF3 gene in the present invention is not particularly limited, but when the expression level of the HSF3 gene in a plant not applied with the DREB2A gene expression promoter of the present invention is 100, the expression of the DREB2A gene of the present invention
  • the expression level of the HSF3 gene in the plant to which the promoter is applied is, for example, 90 or less, preferably 80 or less, preferably 75 or less.
  • the plant that is the target of promoting the expression of the DREB2A gene according to the present invention is a plant that needs to promote the expression of the gene. For example, there is a possibility of being subjected to high-temperature stress such as the above temperature condition or other stresses. It is a plant cultivated in a certain environment. Specific plant types are as described above.
  • the DREB2A gene expression promoter of the present invention may be any agent that contains allantoin and has an action of promoting the expression of the DREB2A gene in plants, and may be allantoin itself, or allantoin and other components may be used. It may be a combined allantoin-containing composition.
  • the DREB2A gene expression promoter of the present invention can contain an effective amount of allantoin that promotes the expression of the DREB2A gene in plants.
  • the DREB2A gene expression promoter of the present invention can also contain an effective amount of allantoin that suppresses the expression of the HSF3 gene in plants.
  • the DREB2A gene expression promoter of the present invention can be in any shape such as solid or liquid.
  • the DREB2A gene expression promoter of the present invention is an allantoin-containing composition
  • the same allantoin-containing composition as described for the high-temperature stress resistance improver can be used.
  • a method of applying the DREB2A gene expression promoter of the present invention to plants plants such as plant roots, stems, leaves, etc., in which the DREB2A gene expression promoter of the present invention or the allantoin released from the DREB2A gene expression promoter of the present invention is used.
  • the method is not particularly limited as long as it is a method capable of contacting the body, and the DREB2A gene expression promoter of the present invention may be applied directly to the plant body, or cultivation of soil or the like to which the plant body has been established.
  • the DREB2A gene expression promoter of the present invention may be applied to the carrier.
  • the DREB2A gene expression promoter of the present invention can be applied to a plant so that an effective amount of allantoin that promotes the expression of the DREB2A gene in the plant is applied.
  • the DREB2A gene expression promoter of the present invention can be applied to a plant so that an effective amount of allantoin that suppresses the expression of the HSF3 gene in the plant is applied.
  • the timing of applying the DREB2A gene expression promoter of the present invention to plants is not particularly limited, but preferably, the DREB2A gene expression promoter of the present invention is applied to plants before being subjected to stress (for example, high temperature stress).
  • the DREB2A gene expression promoter of the present invention promotes the expression of the DREB2A gene.
  • stress for example, high temperature stress.
  • the DREB2A gene expression promoter of the present invention promotes the expression of the DREB2A gene.
  • the DREB2A gene expression promoter of the present invention it is presumed that the expression of various stress resistance genes is induced. For this reason, it is estimated that the plant to which the DREB2A gene expression promoter of the present invention was applied in advance before being stressed is in a state in which stress tolerance has been improved in advance, and the survival rate when stressed thereafter is high.
  • the time point at which the DREB2A gene expression promoter of the present invention is applied to the plant is T1
  • the time point at which the DREB2A gene expression promoter of the present invention is applied to the plant is T1
  • the time point at which the plant begins to be exposed to the stress is T2
  • the time from T1 to T2 is preferably 0.5 to 10 days, preferably 0.5 to 9 days, preferably 0.5 to 8 days, preferably 0.5 to 7 days, preferably Is 0.5-6 days, preferably 0.5-5 days, preferably 0.5-4 days, preferably 0.5-3 days, preferably 0.5-2 days, preferably 0.5-days. 1.5 days.
  • the survival rate after exposure to the stress is high.
  • the DREB2A gene expression promoter of the present invention may be applied N times (N is 2 or more) to the plant before receiving the stress. In this case, each time point T1 n (n Is preferably an integer of 1 to N) to T2 respectively.
  • the DREB2A gene refers to a database (http://www.ncbi.nlm.nih.gov/) provided by NCBI (National Center for Biotechnology Information, National Center for Biotechnology) and other databases.
  • a gene comprising a base sequence encoding a polypeptide annotated as responsive element-binding protein 2A or dehydration-responsive element-binding protein 2A-like, and a base encoding a polypeptide having a function homologous to the polypeptide refers to a gene that contains a sequence.
  • a partial base sequence from the 189th base to the 1196th base in the base sequence shown in SEQ ID NO: 1 is a region encoding the DREB2A polypeptide.
  • nucleotide sequence encoding a polypeptide having a function homologous to a polypeptide annotated as dehydration-responsive-element-binding protein 2A or dehydration-responsive element-binding protein 2A-like is included. Gene expression may be promoted.
  • Examples of the gene containing a base sequence encoding a polypeptide having a function homologous to the polypeptide annotated as dehydration-responsive element-binding protein 2A or dehydration-responsive element-binding protein 2A-like for example, (1) 1 or several, for example, 1 to 20, preferably 1 in the amino acid sequence of the Arabidopsis DREB2A polypeptide encoded by the partial base sequence from the 189th base to the 1196th base in the base sequence shown in SEQ ID NO: 1.
  • a gene comprising a base sequence encoding a polypeptide having a function homologous to the Arabidopsis DREB2A polypeptide, or (2) 60% or more, preferably 70% or more, preferably the amino acid sequence of the Arabidopsis DREB2A polypeptide 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more, preferably Properly comprises an amino acid sequence with a sequence identity of 98% or more, and, the Arabidopsis DREB2A gene comprising a nucleotide sequence encoding a polypeptide having a polypeptide homologous to function but are not limited to these.
  • sequence identity of amino acid sequences can be determined using methods well known to those skilled in the art, sequence analysis software, and the like. For example, when the amino acid sequence of the Arabidopsis DREB2A polypeptide is aligned with the two amino acid sequences by inserting gaps as necessary so that the degree of coincidence between the amino acid sequence and other amino acid sequences is maximized, Refers to the ratio (%) of the number of matched amino acid residues to the number of amino acid residues (including gap number when gaps are inserted), and can be determined using a protein search system by BLAST or FASTA (Karlin, S., et al., 1993, Proceedings of the National, Academic, Sciences, USA, 90, p.
  • the HSF3 gene refers to heat stress in a database (http://www.ncbi.nlm.nih.gov/) provided by NCBI (National Center for Biotechnology Information, National Center for Biotechnology) and other databases.
  • a gene comprising a base sequence encoding a polypeptide annotated as transcription factor A-1b or heat stress transcription factor A-1b-like, and a base sequence encoding a polypeptide having a function homologous to the polypeptide. Refers to the gene containing.
  • a gene containing a base sequence encoding a polypeptide having a function homologous to a polypeptide annotated as heat stress transcription factor A-1b or heat stress transcription factor A-1b-like Expression may be suppressed.
  • Examples of the gene containing a base sequence encoding a polypeptide having a function homologous to the polypeptide annotated as heat stress transcription factor A-1b or heat stress transcription factor A-1b-like (1) In the amino acid sequence of the Arabidopsis thaliana HSF3 polypeptide encoded by the partial base sequence from the 174th base to the 1619th base in the base sequence shown in SEQ ID NO: 2, one or several, for example, 1 to 20, preferably 1, -15, preferably 1-10, preferably 1-5, preferably 1-3, preferably 1 or 2, comprising amino acid sequences substituted, deleted, inserted and / or added And a gene comprising a base sequence encoding a polypeptide having a function homologous to the Arabidopsis thaliana HSF3 polypeptide, or (2) 60% or more, preferably 70% or more, preferably the amino acid sequence of the Arabidopsis thaliana HSF3 polypeptide 80% or more, preferably 85% or more, preferably 90% or more, preferably
  • aln-1 35S: ALN The (3) complement of the allantoinase gene-disrupted strain (aln-1 35S: ALN) is obtained by converting a DNA encoding the entire length of allantoinase derived from wild-type Arabidopsis thaliana into the above-mentioned aln-1 It was obtained by introduction.
  • a specific preparation method is as described in Non-Patent Document 4.
  • One petri dish of 1 / 2MS solid medium was radially divided into three sections, and nine seeds (one petri dish total of 27 grains) were sown in each section for each line (see FIG. 1A).
  • the petri dish was placed in a clean bench with the lid open, the water around the seeds was evaporated (about 20-30 minutes), and the petri dish was sealed with surgical tape.
  • Each petri dish was wrapped with aluminum foil one by one and subjected to low temperature treatment (4 ° C.) for 2 days to overcome sleep.
  • the cells were transferred to a culture room and cultured for 7 days under conditions of 22 ° C.
  • Results A photograph of each petri dish after treatment under each heat shock condition and growth for 1 week is shown in FIG. 1B.
  • the arrangement of plant lines in each petri dish shown in FIG. 1B is as shown in FIG. 1A.
  • the numerical value in the petri dish indicates (number of surviving seedlings) / (number of germinated seedlings) in each section.
  • Fig. 2 shows the survival rate under each heat shock condition.
  • the survival rate shown in FIG. 2 is the result of calculating the average value in two tests by obtaining the survival rate (%) in each condition based on the survival number result shown in FIG. 1B.
  • a polypot (integrated with a balance dish) was placed on a bat and placed in an incubator at 22 ° C., 10000 Lux, 12 hours light period, 12 hours dark period, and cultivation was started. Let the cultivation start date be the 0th day of sowing. On the 5th day of sowing, germination was confirmed. On the 6th day of sowing, 3 individuals (1 plant per site) were left in each pot, and the remaining plants were thinned out.
  • water zone tap water
  • allantoin zone 1 mM allantoin aqueous solution
  • 40 ml / pot was applied to each experimental zone.
  • 40 ml / pot of tap water was supplied to all pots.
  • each bat was placed in a thermostatic chamber and exposed to 45 ° C. control for 1 hour, 1.5 hours, and 2 hours to give high temperature stress.
  • the soil in the pot was kept in sufficient moisture before and after the high temperature stress. After completion of the high temperature stress treatment, it was returned to the incubator at 22 ° C. and cultivation was continued.
  • 40 ml / pot of water was supplied. Further, 40 ml / pot of water was supplied at intervals of 2 days.
  • survival rate indicates the ratio of the surviving plant to the number of plants subjected to heat shock treatment.
  • individuals with physiological disorders whitening, wilting, leaf winding
  • FIG. 3 shows a photograph of the plant on the third day after heat shock treated at 45 ° C. for 1.5 hours.
  • the upper part of FIG. 3 is a photograph of a plant in the allantoin area (survival rate 33.3%), and the lower part of FIG. 3 is a photograph of the plant in the water area (survival rate 0%).
  • a polypot (integrated with a balance dish) was placed on a bat and placed in an incubator at 22 ° C., 10000 Lux, 12 hours light period, 12 hours dark period, and cultivation was started. Let the cultivation start date be the 0th day of sowing.
  • the survival rate on the seventh day after the heat shock was 33.3% in the water zone and 100% in the allanto-in zone after 1 hour treatment. From the above result, the high temperature stress tolerance imparting effect by application of allantoin was confirmed.
  • survival rate indicates the ratio of the surviving plant to the number of plants subjected to heat shock treatment.
  • individuals with physiological disorders whitening, wilting, leaf winding
  • FIG. 4 shows a photograph of the plant on the seventh day after heat shock treated at 45 ° C. for 1 hour.
  • the upper part of FIG. 4 is a photograph of a plant body in the allantoin area (survival rate 100%), and the lower part of FIG. 4 is a photograph of the plant body in the water area (survival rate 33.3%).
  • microarray raw data are registered and published under the accession number of NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). These microarray data were standardized by a parametric method using a three-parameter lognormal distribution model using the SuperNORM service of Skylight Baotech Co., Ltd., and the expression level of each gene was converted into a z-score (see Konishi above) , T. (2004)). This standardization work enables comparison of gene expression levels between different microarrays. Standardized data is registered with the GSE73841 accession number.
  • HSF3 heat shock transcription factor 3 gene
  • the circular accommodating portion in plan view is divided into two semicircular sections by one partition wall extending in the diameter direction.
  • One compartment of the petri dish contains an allantoin-free 1 / 2MS solid medium, the other compartment contains an allantoin-added 1 / 2MS solid medium, and each seed contains 15 seeds (30 dishes in total). (See FIG. 5A).
  • the petri dish was placed in a clean bench with the lid open, the water around the seeds was evaporated (about 20-30 minutes), and the petri dish was sealed with surgical tape. (7) Each petri dish was wrapped with aluminum foil one by one and subjected to low temperature treatment (4 ° C.) for 2 days to overcome sleep. (8) The cells were transferred to a culture room and cultured for 7 days under conditions of 22 ° C. and long days (light irradiation for 16 hours under fluorescent lamps; 0.07 mmol photons m ⁇ 1 s ⁇ 1 ). (9) The 7-day-old plant grown aseptically according to the above (8) was placed in an incubator set at 45 ° C. in advance, and heat shock was applied for 105 minutes in the dark.
  • Results A photograph of each petri dish after treatment under each heat shock condition and growth for 1 week is shown in FIG. 5B.
  • the arrangement of plant lines in each petri dish shown in FIG. 5B is as shown in FIG. 5A.
  • the numerical value in the petri dish indicates (number of surviving seedlings) / (number of germinated seedlings) in each section.
  • Fig. 6 shows the survival rate under each heat shock condition.
  • the survival rate (%) in each condition was obtained based on the result of the survival number shown in FIG. 5B, and the survival rate was similarly obtained for another single test not shown in the photograph. It is the result of calculating the rate (%) and calculating the average value in the two tests.

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Abstract

L'objectif de la présente invention consiste à fournir un moyen permettant d'améliorer la résistance à haute température dans une plante. L'invention concerne un agent permettant d'améliorer la résistance d'une plante aux températures élevées, ledit agent comprenant de l'allantoïne en tant que principe actif. L'invention concerne également un procédé permettant d'améliorer la résistance d'une plante aux températures élevées, ledit procédé comprenant une étape consistant à appliquer l'agent susmentionné permettant d'améliorer la résistance de la plante à une température élevée.
PCT/JP2016/089061 2016-01-29 2016-12-28 Agent d'amélioration de la résistance d'une plante aux températures élevées, procédé d'amélioration de la résistance aux températures élevées, agent de suppression de blanchiment, et un promoteur d'expression de gène dreb2a WO2017130630A1 (fr)

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CN201680080335.6A CN108882712B (zh) 2016-01-29 2016-12-28 植物的高温胁迫抗性提高剂、提高高温胁迫抗性的方法、白化抑制剂、以及dreb2a基因表达促进剂
EP16888214.0A EP3409116B1 (fr) 2016-01-29 2016-12-28 Utilisation de l'allantoïne pour améliorer la résistance aux températures élevées des plantes
ES16888214T ES2955007T3 (es) 2016-01-29 2016-12-28 Uso de alantoína para potenciar la resistencia a altas temperaturas en plantas
US15/977,890 US10182569B2 (en) 2016-01-29 2018-05-11 Composition comprising allantoin and method of applying allantoin to a plant
US16/034,010 US20180310565A1 (en) 2016-01-29 2018-07-12 Composition comprising allantoin and method of applying allantoin to a plant

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CN109206496A (zh) * 2018-11-19 2019-01-15 中国科学院植物研究所 蛋白质GhFLS1在调控植物耐热性中的应用
WO2019216302A1 (fr) * 2018-05-11 2019-11-14 株式会社カネカ Procédé permettant de cultiver des plantes et accélérateur de coloration pour des baies de raisin
WO2023171639A1 (fr) * 2022-03-07 2023-09-14 株式会社カネカ Composition agricole et procédé de culture de plantes l'utilisant
WO2024071065A1 (fr) * 2022-09-26 2024-04-04 株式会社カネカ Composition de revêtement de graine de plante

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CN111154800A (zh) * 2020-03-11 2020-05-15 中国农业科学院作物科学研究所 水稻OsRNCR基因及其编码蛋白在增强植物耐盐性中的应用

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US20180310565A1 (en) * 2016-01-29 2018-11-01 Kaneka Corporation Composition comprising allantoin and method of applying allantoin to a plant
WO2019216302A1 (fr) * 2018-05-11 2019-11-14 株式会社カネカ Procédé permettant de cultiver des plantes et accélérateur de coloration pour des baies de raisin
JPWO2019216302A1 (ja) * 2018-05-11 2021-05-13 株式会社カネカ 植物を栽培する方法及びブドウ果実の着色促進剤
JP7329506B2 (ja) 2018-05-11 2023-08-18 株式会社カネカ 植物を栽培する方法及びブドウ果実の着色促進剤
CN109206496A (zh) * 2018-11-19 2019-01-15 中国科学院植物研究所 蛋白质GhFLS1在调控植物耐热性中的应用
CN109206496B (zh) * 2018-11-19 2020-11-17 中国科学院植物研究所 蛋白质GhFLS1在调控植物耐热性中的应用
WO2023171639A1 (fr) * 2022-03-07 2023-09-14 株式会社カネカ Composition agricole et procédé de culture de plantes l'utilisant
WO2024071065A1 (fr) * 2022-09-26 2024-04-04 株式会社カネカ Composition de revêtement de graine de plante

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