JP7156979B2 - Functional peptides with antibacterial activity against plant pathogens - Google Patents

Description

The present invention relates to a functional peptide that exhibits antibacterial activity against plant pathogenic fungi such as plant pathogenic filamentous fungi, an agricultural chemical containing the functional peptide, and a method for controlling plant pathogenic fungi using the functional peptide.

Reduced crop yields due to plant diseases contribute to the food problem. In order to increase crop yields, it is necessary to control plants from phytopathogenic fungi with fungicides. BACKGROUND ART In recent years, so-called environment-conserving agriculture, in which the amount of pesticides and chemical fertilizers containing chemical substances as main ingredients used has been reduced, has been promoted. Therefore, it is desired to develop substances suitable for environment-conserving agriculture in place of chemical substances.

Such substances include peptidic substances consisting of 100 or less amino acids. Among them, defensins are known as plant-derived peptides having antibacterial activity. Defensins are cysteine-rich basic proteins that have been reported in many plant species. However, defensins need to maintain their higher-order structures in order to exhibit antibacterial activity, and since they are relatively long peptides of 40 amino acids or more, they are difficult and expensive to synthesize. Non-Patent Document 1 discloses the results of synthesizing partial peptides of rice-derived defensins and examining their antibacterial activities. However, the defensin partial peptides disclosed in Non-Patent Document 1 have only been shown to be effective against blast disease, wilt disease, and rice bakanae disease.

Moreover, Patent Document 1 discloses that complex disease resistance can be imparted to plants by introducing defensin genes derived from cabbage and Komatsuna belonging to the Brassicaceae family. Patent Document 2 discloses that an Arabidopsis thaliana-derived peptide having antibacterial activity was isolated. Patent Document 3 discloses a peptide having an antibacterial activity against rice blast fungi extracted from Pleurotus cornucopia, which is one of edible mushrooms. Patent Document 4 discloses 10 to 16 partial peptides having antibacterial activity designed from rice-derived defensin-like proteins having antibacterial activity. Patent Document 5 discloses that a peptide having antibacterial activity derived from Arabidopsis thaliana was isolated. Patent Document 6 discloses an antibacterial peptide consisting of 40 to 66 amino acids prepared from the body fluid of larvae of the Formosan beetle. Patent document 7 shows a method for obtaining antimicrobial peptides against plant pathogens based on defensins by DNA shuffling.

Japanese Patent Application Laid-Open No. 2004-329215 Japanese Patent Publication No. 2006-512052 Patent No. 4257119 Patent No. 5988200 Patent No. 4445855 International publication WO2003/033532 U.S. Pat. No. 8,865,967

J.Pestic.Sci.42(4), 172-175, 2017

However, short functional peptides that have antibacterial activity against a wide variety of plant pathogens and that are easy to synthesize have not been known. Therefore, in view of the actual situation described above, the present invention uses a functional peptide having antibacterial activity against a wide variety of plant pathogens, a nucleic acid encoding the functional peptide, an agricultural chemical containing the functional peptide, and the functional peptide. An object of the present invention is to provide a method for controlling plant pathogens.

As a result of intensive studies by the present inventors in order to achieve the above-mentioned purpose, peptides having antibacterial activity against a wide variety of plant pathogens were identified from peptides specifically expressed in the ductal fluid of tomatoes inoculated with wilt fungus. As a result, the present invention has been completed.

The present invention includes the following.
(1) Amino acid sequence: consisting of MDFHDVWRSNIQDVRKLALETSKCVLMGPSLFGRVKFICFG (SEQ ID NO: 1), or consisting of an amino acid sequence having 85% or more identity to the amino acid sequence, or 5 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 1 A functional peptide consisting of a sequence.
(2) The continuous amino acid sequence of 5 or more is an amino acid sequence in the range of 19 to 41 amino acid residues counted from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. functional peptide.
(3) The functional peptide according to (1), which consists of LETSKCVLMGPSLFGRVKFICFG (SEQ ID NO: 2).
(4) A nucleic acid encoding the functional peptide according to any one of (1) to (3) above.
(5) A recombinant vector having the nucleic acid described in (4) above.
(6) A transformant having the recombinant vector described in (5) above.
(7) The transformant according to (6), which is a plant cell or plant transformed with the recombinant vector.
(8) A composition exhibiting a bactericidal or antibacterial action against plant pathogenic bacteria, comprising the functional peptide according to any one of (1) to (3) above.
(9) An agricultural chemical comprising the functional peptide according to any one of (1) to (3) above.
(10) The agricultural chemical according to (9), which is for sterilization or antibacterial use against plant pathogens.
(11) The plant pathogens include Botrytis cinerea, Colletotrichum destructivum, Colletotrichum orbiculare, Fusarium oxysporum and Pyricularia oryzae. The agricultural chemical according to (10), which is one or more phytopathogenic filamentous fungi selected from the group.

The functional peptide according to the present invention has excellent antibacterial activity against a wide variety of plant pathogens, and can be easily synthesized. Therefore, the functional peptide according to the present invention can be used, for example, as a composition or an agricultural chemical having a bactericidal action or antibacterial action against plant pathogenic bacteria.

Moreover, by using a nucleic acid encoding a functional peptide according to the present invention, the functional peptide can be expressed in a host. By expressing the above functional peptides in plant cells or plants, resistance to a wide variety of plant pathogenic fungi is improved.

Fig. 10 is a photograph showing the results of inoculating tomato leaves with TsORF17-2 together with Botrytis cinerea. Fig. 10 is a photograph showing the results of inoculating Chinese cabbage leaves with TsORF17-2 together with Colletotrichum destructivum.

<Functional peptide>
The functional peptide according to the present invention consists of the amino acid sequence MDFHDVWRSNIQDVRKLALETSKCVLMGPSLFGRVKFICFG shown in SEQ ID NO: 1, or consists of an amino acid sequence having 85% or more identity to the amino acid sequence. Here, the amino acid sequence shown in SEQ ID NO: 1 was obtained by comprehensively analyzing peptides contained in ductal fluid collected from tomato infected with wilt disease and ductal fluid collected from uninfected tomato. It was identified as a peptide with antibacterial activity against pathogenic bacteria. That is, the functional peptide according to the present invention has at least the function of having antibacterial activity against plant pathogenic bacteria. Therefore, the functional peptide according to the present invention can be used as an agricultural chemical or an agricultural chemical composition based on its antibacterial activity against plant pathogens.

An amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 1 specifically means that 1 to 4 amino acids are deleted, substituted, or deleted from the amino acid sequence of SEQ ID NO: 1 It means an added amino acid sequence. The functional peptide according to the present invention preferably has 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1. Identity is more preferred. An amino acid sequence having this range of identity with the amino acid sequence shown in SEQ ID NO: 1 has a high probability of exhibiting antibacterial activity against plant pathogenic bacteria, like a peptide consisting of the amino acid of SEQ ID NO: 1.

In particular, the amino acid sequence in which 1 to 4 amino acids are deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 1 is not particularly limited, but the positions where 1 to 4 amino acids are deleted, substituted, or added is preferably near the N-terminus. More specifically, the position where 1 to 4 amino acids are deleted, substituted or added is the 1st to 18th range, preferably 1st to 15th, counting from the N-terminus in the amino acid sequence shown in SEQ ID NO: 1. , more preferably in the 1st to 10th range. Deleting, substituting, or adding 1 to 4 amino acids within this range can maintain high antibacterial activity against plant pathogenic bacteria.

An amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1 may be obtained using a mutation introduction kit (for example, Mutant-K (manufactured by Takara Bio Inc.) or Mutant-G (manufactured by Takara Bio Inc.) using site-directed mutagenesis. ), etc., or by introducing a desired mutation into a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 1 using Takara Bio Inc.'s LA PCR in vitro Mutagenesis series kit. can be done.

By the way, the functional peptide according to the present invention is not limited to the one consisting of the amino acid sequence shown in SEQ ID NO: 1 (41 amino acid residues in length), but may consist of 5 or more consecutive amino acids in the amino acid sequence. There may be. A peptide consisting of 5 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 1 may be hereinafter referred to as a partial peptide. The partial peptide is not particularly limited, but a continuous 5 to 25 amino acid sequence, preferably a 10 to 25 amino acid sequence, more preferably an 18 to 23 amino acid sequence selected from the amino acid sequence of SEQ ID NO: 1, and further Peptides consisting of 23 amino acids are preferred.

In particular, the partial peptide is preferably selected from the range of 19 to 41 amino acid residues counted from the N-terminus of the amino acid sequence shown in SEQ ID NO:1. A partial peptide having an amino acid sequence within this range can exhibit superior antibacterial activity.

More specifically, the partial peptide is preferably LETSKCVLMGPSLFGRVKFICFG (SEQ ID NO: 2). The partial peptide can exhibit particularly excellent antibacterial activity against plant pathogenic bacteria.

The functional peptides (including the partial peptides described above) according to the present invention can be easily produced by conventionally known techniques. Methods for synthesizing peptides include, for example, solid-phase synthesis and liquid-phase synthesis. In the solid-phase synthesis method, the Fmoc method (fluorenylmethyloxycarbonyl method), the tBoc method (t-butyloxycarbonyl method), or the like can be applied to synthesize a functional peptide having a desired amino acid sequence.

In addition, the functional peptide according to the present invention can also be produced by producing a nucleic acid encoding the amino acid sequence and by genetic engineering techniques using the nucleic acid. Specifically, for example, a nucleic acid encoding the functional peptide described above is chemically synthesized, incorporated into an expression vector, and the obtained expression vector is introduced into an appropriate host cell to obtain a transformant. After culturing this transformant to produce a functional peptide, it may be recovered from the transformant and/or the medium.

The expression vector is not particularly limited as long as it has a nucleic acid encoding a functional peptide and can express the functional peptide in any host cell. Expression vectors include, for example, plasmid vectors, virus vectors and the like. The host cell is not particularly limited, and bacteria such as Escherichia coli and Bacillus subtilis, and fungi such as yeast and filamentous fungi can be used. Animal cells such as insect cells and mammalian cells may also be used as host cells. For example, when E. coli is used as a host cell, an expression vector containing at least a promoter region, an initiation codon, a nucleic acid encoding the functional peptide of the present invention, a termination codon, a terminator region and a replication origin can be used. .

The functional peptide according to the present invention can be purified after culturing the transformant as described above and recovering from the transformant. Methods for isolating and purifying functional peptides are not particularly limited, and examples include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrolysis. Methods that use molecular weight differences such as electrophoresis, methods that use charge such as ion exchange chromatography and hydroxylapatite chromatography, methods that use specific affinity such as affinity chromatography, and hydrophobic methods such as reversed-phase high-performance liquid chromatography Examples include a method using a difference in sex and a method using a difference in isoelectric point such as isoelectric focusing.

Moreover, the functional peptide according to the present invention can be produced by a transformant with a histidine tag, a Flag tag, or the like added, for example. In this case, the functional peptide can be isolated and purified more easily by using a substance that has affinity for the tag.

Furthermore, the functional peptide according to the present invention can be synthesized using a so-called cell-free protein synthesis system. In a cell-free protein synthesis system, for example, extracts from Escherichia coli, rabbit reticulocytes, and wheat germ can be used.

<Antibacterial activity in functional peptide>
The functional peptide according to the present invention has antibacterial activity against a wide variety of plant pathogenic bacteria. Here, the term "antibacterial activity" includes both an activity to prevent plant pathogenic fungi infection and a fungicidal activity against infected plant pathogenic fungi. Plant pathogenic filamentous fungi can be widely exemplified as plant pathogenic fungi for which the functional peptide according to the present invention exhibits antibacterial activity. More specifically, plant pathogenic fungi include plant pathogenic filamentous fungi belonging to the genus Botrytis, plant pathogenic filamentous fungi belonging to the genus Colletotrichum, plant pathogenic filamentous fungi belonging to the genus Fusarium, and plant pathogenic filamentous fungi belonging to the genus Pyricularia. .

Plant pathogenic filamentous fungi belonging to the genus Botrytis include Botrytis aclada, Botrytis byssoidea, Botrytis cinerea, Botrytis convoluta, Botrytis diospyri, Botrytis elliptica, Botrytis fabae, Botrytis galanthina, Botrytis gladiolorum, Botrytis paeoniae, Botrytis polyblastis, Botrytis sp., and Botrytis squamosa. and Botrytis tulipae.

Plant pathogenic filamentous fungi belonging to the genus Colletotrichum include Colletotrichum actinidiicola, Colletotrichum acutatum, Colletotrichum aenigma, Colletotrichum ampelopsidis, Colletotrichum belamcandium, Colletotrichum boninense sensu lato, Colletotrichum capsici, Colletotrichum carthami, Colletoanstrichum caudatum, Colletotrichum chrystotrichum, Colletotrichum colletotrichum, Colletotrichum colletotrichum coletotrichum. coffeanum、Colletotrichum corchori、Colletotrichum crassipes、Colletotrichum cypripedii、Colletotrichum daphnicola、Colletotrichum dematium、Colletotrichum destructivum、Colletotrichum durionis、Colletotrichum echinochloae、Colletotrichum elasticae、Colletotrichum fatsiae、Colletotrichum fioriniae、Colletotrichum fuscum、Colletotrichum gloeosporioides、Colletotrichum godetiae、Colletotrichum graminicola、Colletotrichum hibisci、 Colletotrichum higginsianum, Colletotrichum horii, Colletotrichum hydrangeae, Colletotrichum kahawae, Colletotrichum karstii, Colletotrichum koyasuensis, Colletotrichum lappae, Colletotrichum liliacearum, Colletotrichum lilii, Colle totrichum lindemuthianum、Colletotrichum malvarum、Colletotrichum medicaginis-denticulatae、Colletotrichum metake、Colletotrichum moricola、Colletotrichum morinum、Colletotrichum musae、Colletotrichum nigrum、Colletotrichum nymphaeae、Colletotrichum orbiculare、Colletotrichum panacicola、Colletotrichum paniculatae、Colletotrichum pehkinense、Colletotrichum phaseolorum、Colletotrichum sansevieriae、Colletotrichum sasicola、 Colletotrichum siamense, Colletotrichum sophorae-japonicae, Colletotrichum spaethianum, Colletotrichum spinaciae, Colletotrichum sublineolum, Colletotrichum tabacum, Colletotrichum theobromicola, Colletotrichum trichellum, Colletotrichum trifolii, Colletotrichum tropicale, Colletotrichum truncatum, Colletotrichum truncatum, Colletotrichum villosum and Colletotrichum villosum.

Fusarium acuminatum, Fusarium ananatum, Fusarium anguioides, Fusarium arthrosporioides, Fusarium asiaticum, Fusarium avenaceum, Fusarium brasiliense, Fusarium commune, Fusarium conglutinans var. betae, Fusarium cuneirostrum, Fusarium decemcellulare, and Fusarium dimerum var. dimerum, Fusarium foetens, Fusarium fujikuroi, Fusarium graminearum, Fusarium guttiforme, Fusarium lactis, Fusarium lagenariae, Fusarium lateritium, Fusarium merismoides, Fusarium oxysporum, Fusarium pallidoroseum, Fusarium pallidum, Fusarium phaseoli, Fusarium phyllum, Fusarium poae, Fusarium prophiliferens, Fusarium proliferatum , Fusarium ricini, Fusarium roseum, Fusarium solani, Fusarium striatum, Fusarium subglutinans and Fusarium verticillioides.

Plant pathogenic filamentous fungi belonging to the genus Pyricularia include Pyricularia grisea, Pyricularia higginsii, Pyricularia oryzae, Pyricularia panici and Pyricularia zingiberis.

In addition to these, plant pathogens include, for example, bacteria belonging to the genus Alternaria, bacteria belonging to the genus Cladosporium, bacteria belonging to the genus Claviceps, bacteria belonging to the genus Sclerotinia, bacteria belonging to the genus Septoria, bacteria belonging to the genus Pseudoperonospora, and the genus Puccinia. can include bacteria belonging to

In particular, plant pathogenic filamentous fungi with high antibacterial activity by the functional peptide according to the present invention include Botrytis cinerea, Colletotrichum destructivum, Colletotrichum orbiculare, Fusarium oxysporum and Pyricularia oryzae (synonymous with Magnaporthe oryzae).

Furthermore, the functional peptide according to the present invention has antibacterial activity against not only plant pathogenic filamentous fungi but also plant pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. Tomato.

Plant pathogenic bacteria belonging to the genus Xanthomonas include Xanthomonas campestris pv. malloti, Xanthomonas axonopodis pv. phaseoli, Xanthomonas alfalfae pv. alfalfae, Xanthomonas arboricola pv. oryzicola, Xanthomonas axonopodis pv. phaseoli, Xanthomonas translucens pv. translucens, Xanthomonas arboricola, Xanthomonas campestris pv. raphani, Xanthomonas campestris pv. Xanthomonas axonopodis pv. manihotis、Xanthomonas arboricola pv. juglandis、Xanthomonas campestris pv. nigromaculans、Xanthomonas albilineans、Xanthomonas axonopodis pv. vasculorum、Xanthomonas campestris、Xanthomonas cucurbitae、Xanthomonas axonopodis pv. glycines、Xanthomonas campestris pv. cannabis、Xanthomonas translucens pv. phleipratensis , Xanthomonas theicola, Xanthomonas vesicatoria, Xanthomonas euvesicatoria, Xanthomonas hortorum pv. carotae, Xanth omonas axonopodis pv. allii、Xanthomonas arboricola pv. celebensis、Xanthomonas axonopodis pv. ricini、Xanthomonas hortorum pv. pelargonii、Xanthomonas translucens pv. cerealis、Xanthomonas campestris pv. mangiferaeindicae、Xanthomonas vasicola pv. holcicola、Xanthomonas axonopodis pv. lespedezae、Xanthomonas translucens pv. cerealis, Xanthomonas hortorum, Xanthomonas axonopodis pv. Vitians and Xanthomonas citri subsp. Malvacearum.

As described above, the functional peptide according to the present invention has antibacterial activity against a wide variety of plant pathogenic bacteria, and thus can control plant diseases caused by plant pathogenic bacteria exhibiting antibacterial activity. Specifically, plant diseases that can be controlled by the functional peptides of the present invention include, for example, rice diseases such as rice blast, sesame leaf blight, sheath blight, bananae disease, and white leaf blight. . Plant diseases that can be controlled by the functional peptide according to the present invention are not particularly limited, but powdery mildew, Fusarium head blight, rust, snow rot, snow rot, smut, smut, and smut. Smut, catfish smut, eye spot, leaf blight, downy blight, yellow spot, cloud, net spot, leaf spot, spotted leaf disease, seedling blight caused by Rhizoctonia, leopard, southern Rust, gray leaf spot, purpura, black spot, black spot, brown spot, brown spot, sclerotia, anthracnose, summer plague, scarlet rot, powder scab and white mold disease can be mentioned.

<Agrochemicals and Compositions Containing Functional Peptides>
Since the functional peptide according to the present invention has an antibacterial action against a wide variety of plant pathogens, it can be used as an agricultural chemical or an agricultural chemical composition. Here, the agricultural chemical composition means a composition that can be used when producing an agricultural chemical. For example, the functional peptide according to the present invention can be used as a fungicide having a fungicidal action against plant pathogenic fungi, particularly plant pathogenic filamentous fungi. More specifically, the functional peptide compound may be used as an agricultural chemical as it is, but it is usually mixed with a suitable solid carrier, liquid carrier, etc., a surfactant and other formulation aids to form an emulsifiable concentrate, EW formulation, Liquid formulations, suspensions, wettable powders, wettable granules, powders, DL powders, fine granules, fine granules F, granules, tablets, oils, aerosols, flowables, dry flowables, microcapsules, etc. Can be used as a mold.

Examples of solid carriers include starch, activated carbon, soybean flour, wheat flour, wood flour, fish meal, animal and plant powders such as milk powder, talc, kaolin, bentonite, calcium carbonate, zeolite, diatomaceous earth, white carbon, clay, alumina, ammonium sulfate, and urea. and other inorganic powders.

Examples of liquid carriers include water; alcohols such as isopropyl alcohol and ethylene glycol; ketones such as cyclohexanone and methyl ethyl ketone; ethers such as dioxane and tetrahydrofuran; Aromatic hydrocarbons such as tetramethylbenzene, methylnaphthalene, solvent naphtha; Halogenated hydrocarbons such as chlorobenzene; Acid amides such as dimethylacetamide; Esters such as glycerol esters of fatty acids; Nitriles such as acetonitrile; sulfur-containing compounds such as sulfoxide;

Examples of surfactants include alkylbenzenesulfonic acid metal salts, dinaphthylmethanedisulfonic acid metal salts, alcohol sulfates, alkylarylsulfonates, ligninsulfonates, polyoxyethylene glycol ethers, polyoxyethylene alkylaryl ethers, and polyoxyethylene sorbitan monoalkylate.

Other adjuvants include, for example, fixing agents or thickeners such as carboxymethylcellulose, gum arabic, sodium alginate, guar gum, tragacanth gum, and polyvinyl alcohol, antifoaming agents such as metallic soap, fatty acids, alkyl phosphates, silicones, and paraffin. A physical property improver such as a coloring agent can be used.

Various formulations of fungicides or their dilutions are applied by a commonly used application method, i.e., spraying (e.g., spraying, misting, atomizing, dusting, granules, water surface application, box application, etc.) , soil application (eg, mixing, irrigation, etc.), surface application (eg, coating, dressing, coating, etc.), immersion, bait, smoke application, and the like.

Furthermore, it goes without saying that the fungicide containing the functional peptide according to the present invention as an active ingredient is sufficiently effective even when the functional peptide alone is used as an active ingredient. It can be mixed or used together with insecticides, acaricides, nematicides, other fungicides, antiviral agents, attractants, herbicides, plant growth regulators and the like.

The pesticide composed as described above can be applied to plants that are susceptible to plant diseases caused by the plant pathogenic fungi described above. Plants to be applied are not particularly limited, but for example, crops: corn, rice, wheat, barley, rye, oat, sorghum, cotton, soybean, peanut, buckwheat, sugar beet, rape, sunflower, sugarcane, tobacco, solanaceae. Vegetables (eggplant, tomato, green pepper, hot pepper, potato, etc.), cucurbitaceous vegetables (cucumber, pumpkin, zucchini, watermelon, melon, squash, etc.), cruciferous vegetables (radish, turnip, horseradish, kohlrabi, Chinese cabbage, cabbage, mustard greens) , broccoli, cauliflower, etc.), Asteraceous vegetables (burdock, chrysanthemum, artichoke, lettuce, etc.), Liliaceous vegetables (leeks, onions, garlic, asparagus, etc.), Umbelliferous vegetables (carrots, parsley, celery, American bowhu, etc.) , Chenopodiaceous vegetables (spinach, chard, etc.), labiatae vegetables (perilla, mint, basil, etc.), strawberries, sweet potatoes, yam, taro, etc.; Japanese pear, quince, quince, etc.), stone fruits (peach, plum, nectarine, plum, cherry, apricot, prunes, etc.), citrus fruits (unshu mandarin, orange, lemon, lime, grapefruit, etc.), nuts (chestnut, walnut, hazel) , almonds, pistachios, cashew nuts, macadamia nuts, etc.), berries (blueberries, cranberries, blackberries, raspberries, etc.), grapes, persimmons, olives, loquats, bananas, coffee, dates, coconut palms, etc.; , mulberry, jatropha, flowering trees, roadside trees (ash, birch, dogwood, eucalyptus, ginkgo, lilac, maple, oak, poplar, redbud, sycamore, plane tree, zelkova, arborvitae, fir tree, hemlock, juniper, pine, spruce, yew).

Among them, it is preferable to apply the pesticide containing the above functional peptide as an active ingredient to cruciferous plants, cucurbitaceous plants and gramineous plants.

<Transformed plant>
As described above, the functional peptide according to the present invention has antibacterial activity against a wide variety of plant pathogenic bacteria. Therefore, a transgenic plant that highly expresses the functional peptide is characterized by exhibiting resistance to the plant diseases described above. Transformed plants include both transformed plant cells in which a nucleic acid encoding the functional peptide described above has been introduced into predetermined plant cells so as to be expressible, and transformed plants in which the transformed plant cells are regenerated into plants. Meaning. The transgenic plant according to the present invention has improved resistance to the above-described plant pathogenic fungi by expressing the above-described functional peptide.

More specifically, transformed plants include, but are not limited to, corn, rice, wheat, barley, rye, oat, sorghum, cotton, soybean, peanut, buckwheat, sugar beet, rapeseed, sunflower, sugarcane, tobacco, and eggplant. Cucurbitaceous vegetables (eggplant, tomato, green pepper, hot pepper, potato, etc.), Cucurbitaceous vegetables (cucumber, pumpkin, zucchini, watermelon, melon, squash, etc.), Cruciferous vegetables (radish, turnip, horseradish, kohlrabi, Chinese cabbage, cabbage, Mustard, broccoli, cauliflower, etc.), Asteraceae vegetables (burdock, chrysanthemum, artichoke, lettuce, etc.), Liliaceae vegetables (green onions, onions, garlic, asparagus, etc.), Umbelliferae vegetables (carrots, parsley, celery, American bowhu, etc.) ), Chenopodiaceous vegetables (spinach, chard, etc.), Labiatae vegetables (perilla, mint, basil, etc.), strawberries, sweet potatoes, yam, taro, etc.; , Japanese pear, quince, quince, etc.), stone fruits (peach, plum, nectarine, plum, cherry, apricot, prunes, etc.), citrus fruits (unshu mandarin, orange, lemon, lime, grapefruit, etc.), nuts (chestnuts, walnuts, Hazel, almonds, pistachios, cashews, macadamia nuts, etc.), berries (blueberries, cranberries, blackberries, raspberries, etc.), grapes, persimmons, olives, loquats, bananas, coffee, dates, coconuts, etc.; trees other than fruit trees: Tea, mulberry, jatropha, flowering trees, roadside trees (ash, birch, dogwood, eucalyptus, ginkgo, lilac, maple, oak, poplar, redbud, sweetberry, plane tree, zelkova, arborvitae, Japanese maple, hemlock, juniper, pine , spruce, yew).

The method for producing a transformed plant according to the present invention is not particularly limited. Generally speaking, a nucleic acid encoding the functional peptide described above is incorporated into an expression vector, and the resulting expression vector is introduced into a plant. Transformed plants can be produced that express the functional peptides described above.

An expression vector is constructed to contain a promoter that enables expression in plants and a nucleic acid that encodes the functional peptide described above. Various conventionally known vectors can be used as the parent vector of the expression vector. For example, a plasmid, phage, cosmid, or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the method of introduction. Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, and pBI-based vectors. In particular, when the method of introducing the vector into the plant body is a method using Agrobacterium, it is preferable to use a pBI-based binary vector. Specific examples of pBI-based binary vectors include pBIG, pBIN19, pBI101, pBI121, and the like.

The promoter is not particularly limited as long as it is capable of expressing the nucleic acid encoding the functional peptide in a plant, and known promoters can be suitably used. In particular, as the promoter, it is preferable to use a constitutive expression promoter that constitutively expresses the downstream gene in the plant body. Such promoters include, for example, cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, nopaline synthase gene promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-bisphosphate carboxylase. - Oxygenase small subunit gene promoter, napin gene promoter and the like can be mentioned. Among these, cauliflower mosaic virus 35S promoter, actin gene promoter or ubiquitin gene promoter can be used more preferably. By using each of the above promoters, any gene can be strongly expressed when introduced into a plant cell.

In addition, promoters that have the function of site-specific expression in plants can also be used. Any conventionally known promoter can be used as such a promoter. Using such a promoter, the functional peptide can be expressed site-specifically.

Furthermore, a gene promoter that induces expression in response to disease can be used as the promoter. Examples of such promoters include plant defensin 1.2 (PDF1.2) gene promoter, pathogenesis-related 1 (PR1) gene promoter, PR2 gene promoter, PR5 gene promoter and the like. Using such a promoter, the functional peptide can be specifically expressed during infection with pathogens.

The expression vector may further contain other DNA segments in addition to the nucleic acid encoding the promoter and the functional peptide. The other DNA segment is not particularly limited, but includes terminators, selection markers, enhancers, base sequences for enhancing translation efficiency, and the like. In addition, the expression vector may further have a T-DNA region. The T-DNA region can enhance the efficiency of gene transfer, particularly when Agrobacterium is used to transfer the above recombinant expression vector into a plant.

The transcription terminator is not particularly limited as long as it functions as a transcription termination site, and may be a known one. For example, specifically, the transcription termination region (Nos terminator) of the nopaline synthase gene, the transcription termination region of cauliflower mosaic virus 35S (CaMV35S terminator), and the like can be preferably used. Among these, the Nos terminator can be used more preferably. By placing a transcription terminator at an appropriate position in the expression vector, it is possible to prevent the occurrence of a phenomenon in which an unnecessarily long transcript is synthesized after introduction into a plant cell.

As a transformant selection marker, for example, a drug resistance gene can be used. Specific examples of such drug-resistant genes include drug-resistant genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol, and the like. Thus, transformed plants can be easily selected by selecting plants that grow in a medium containing the antibiotic.

The construction method of the expression vector is also not particularly limited, and the promoter, the nucleic acid encoding the functional peptide, and, if necessary, the other DNA segments are placed in an appropriately selected parent vector in a predetermined order. It should be introduced so that For example, an expression cassette may be constructed by ligating a nucleic acid encoding the above functional peptide and a promoter (including a transcription terminator, if necessary), and introducing it into a vector. In constructing an expression cassette, for example, the cleavage sites of each DNA segment are provided with mutually complementary protruding ends, and the sequences of the DNA segments can be defined by reacting with a ligation enzyme. When the expression cassette contains a terminator, the sequence may be the promoter, the nucleic acid encoding the functional peptide, and the terminator from upstream. In addition, the types of reagents for constructing the expression vector, that is, the types of restriction enzymes, ligation enzymes, etc., are not particularly limited, and commercially available ones may be appropriately selected and used.

The expression vector described above is introduced into the target plant by common transformation methods. A method for introducing an expression vector into a plant cell (transformation method) is not particularly limited, and an appropriate conventionally known method suitable for the plant cell can be used. Specifically, for example, a method using Agrobacterium or a method of direct introduction into plant cells can be used. Examples of methods for directly introducing expression vectors into plant cells include microinjection, electroporation, polyethylene glycol, particle gun, protoplast fusion, calcium phosphate, and the like.

In addition, if a method of directly introducing DNA into plant cells is adopted, a DNA containing a transcription unit necessary for expression of a gene of interest, such as a promoter or a transcription terminator, and a nucleic acid encoding the above functional peptide is sufficient. and the vector function is not required. Furthermore, even a DNA containing only the coding region of the functional peptide without a transcription unit may be used as long as it can be integrated into the transcription unit of the host and express the target functional peptide.

Examples of plant cells into which the above expression vector or an expression cassette containing a nucleic acid encoding a target functional peptide without an expression vector is introduced include cells of each tissue in plant organs such as flowers, leaves, and roots. , callus, suspension cultured cells, and the like. Here, an appropriate expression vector may be appropriately constructed according to the type of plant body to be produced. good too.

Tumor tissues, shoots, hairy roots, etc. obtained as a result of transformation can be directly used for cell culture, tissue culture or organ culture. plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.) can be administered to the plant body.

As a regeneration method, a method is adopted in which callus-like transformed cells are transferred to a medium containing different types and concentrations of hormones and cultured to form somatic embryos to obtain complete plant bodies. The medium to be used is exemplified by LS medium, MS medium and the like.

In addition, the transformed plant according to the present invention is obtained by introducing an expression vector containing a nucleic acid encoding the above-described functional peptide into a host cell to obtain a transformed plant cell, and transforming the transformed plant from the transformed plant cell. It also means to obtain a plant seed from a transformed plant body obtained by regenerating, and to include progeny plants obtained from the plant seed. To obtain plant seeds from a transformed plant, for example, the transformed plant is collected from a rooting medium, transplanted into a pot containing water-containing soil, grown at a constant temperature, and formed into flowers. and eventually form seeds. Also, in order to produce plants from seeds, for example, when the seeds formed on the transformed plants have matured, they are isolated, sown in water-containing soil, and allowed to grow under constant temperature and illumination. By doing so, plants are produced. Plants produced in this manner express the functional peptides described above, and thus exhibit excellent resistance to the above-described plant pathogenic fungi.

EXAMPLES The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

In this example, functional peptides and partial peptides thereof having antibacterial activity against phytopathogenic filamentous fungi were identified in the following order.
1. Drainage collection of wilt-infected/uninfected tomatoes2. Proteome analysis3. Peptide identification in ductal fluid4. Synthesis of functional peptides5. Evaluation of antibacterial activity against phytopathogenic filamentous fungi Below, the above 1. ~ 5. explain in order

<1. Collection of ductal fluid from wilt-infected/non-infected tomatoes>
Tomato cultivar Ponterosa was grown in Kumiai Nippi gardening medium. A tomato seedling with two unfolded true leaves was dug up, and the root was washed with tap water. The roots were immersed in a bud cell suspension (1×10 ⁶ cells/ml) of tomato wilt fungus (Fusarium oxysporum CK3-1 strain) for about 30 seconds, then transplanted into a vinyl pot containing Kumiai Nippi gardening medium. It was grown in a climate chamber (25°C, 14 hours light, 10 hours dark). As a control group, non-inoculated seedlings were grown in the same manner. 7, 9, 11 and 13 days after transplantation, the inoculated seedlings and the non-inoculated seedlings were cut directly under the first true leaf, the pot was turned over, and the cut surface of the stem was placed in a 2.0 ml plastic tube. It was allowed to stand at room temperature for 12 hours, and the duct fluid flowing out from the cut surface was collected. After measuring the volume of the collected liquid, it was filtered through a cellulose filter (Minisart RC15, Sartrium Stedim) and stored at -80°C.

The Bio-Rad DC Protein Assay Kit (Bio-Rad) was used to measure the protein concentration of the ductal fluid. Bovine serum albumin (BSA) was used to create a calibration curve.

<2. Proteome Analysis>
In-gel trypsin digestion of duct fluid proteins
To duct fluid samples (12 μl, about 10 μg) collected from tomato wilt-inoculated and non-inoculated seedlings, 4 μl of 4× sample buffer (Wako Pure Chemical Industries, Ltd.) was added. It was divided and further chopped into pieces of 1 mm square. In-gel digestion was performed by the method of Rosefeld et al. (1992) (Rosenfeld, J., Capdevielle, J., Guillemot, JC and Ferrara, P. (1992). In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203: 173-179).

First, the gel was washed once each with Milli-Q water (ultrapure water) and 50 mM ammonium carbonate/50% acetonitrile, and dried with acetonitrile. After adding 50 mM tris(2-carboxyethyl)phosphine and standing at 60° C. for 10 minutes, 100 mM 2-iodoacetamide was added and left at room temperature for 1 hour. The gel was washed once with milli-Q water (ultrapure water) and 50 mM ammonium carbonate/50% acetonitrile, dried with acetonitrile, and Lys-C/trypsin (0.01 mg/ml, manufactured by Promega) was added to form a gel. was swollen. After adding 20 μl of 50 mM ammonium carbonate to the swollen gel, the mixture was allowed to react at 37° C. for 16 hours. After adjusting the reaction solution to pH=2 using 20% trifluoroacetic acid, it was filtered through PVDF 0.1 μm of Durapore membrane (manufactured by Merck Millipore), concentrated under reduced pressure, and diluted with 0.1% trifluoroacetic acid/2% acetonitrile. Dissolved.

nano LC-MS/MS analysis
For nano-LC-MS/MS analysis, a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) combined with a Dionex U3000 gradient pump (Thermo Fisher Scientific) was used. board. Using a trap column (L-column ODS, 300 μm I.D.×5 mm, 5 μm particle size, Chemicals Evaluation and Research Institute) and an analysis column (NTCC-360, 100 μm I.D.×125 mm, 3 μm particle size, Nikkyo Technos) Therefore, mobile phase A (0.5% acetic acid) and mobile phase B (80% acetonitrile containing 0.5% acetic acid) were used. The flow rate was 0.5 μl/min and the gradient was 5% B to 35% B in 100 min, 35% B to 100% B in 1 min, 100% B in 3 min, 100% B in 1 min. 5% B and finally 5% B for 10 minutes.

<3. Identification of peptides in ductal fluid>
Proteins detected by nano LC-MS/MS analysis were analyzed using Proteome Discoverer 2.0.0.802 (manufactured by Thermo Fisher Scientific). Tomato sORF database (ITAG2.4_gene_models_sORF), tomato protein database (ITAG2.4_protein), and tomato wilt fungus protein database (fusarium_oxysporum_f._sp._lycopersici_4827_2_protein) were used as databases. As a result, 4 peptides (designated as TsORF6, 11, 19, 29) were confirmed to be expressed specifically in non-inoculated tomatoes, and 4 peptides (designated as TsORF8, 15, 18, 30) were specifically expressed in inoculated tomatoes. , 6 peptides (named TsORF10, 16, 17, 21, 24, 25) that were commonly expressed in non-inoculated and inoculated tomatoes were found.

The 14 types of peptides identified in this example are not registered in the tomato protein database (ITAG2.4_protein) and the tomato wilt fungus protein database (fusarium_oxysporum_f._sp._lycopersici_4827_2_protein), and the tomato sORF database (ITAG2.4_gene_models_sORF) ) is a peptide registered only in This tomato sORF database is disclosed in sORF finder: a program package to identify small open reading frames with high coding potential K Hanada, K Akiyama, T Sakurai, T Toyoda, K Shinozaki, SH Shiu Bioinformatics 26 (3), 399-400 A database containing information on small open reading frames (sORFs) encoding peptides of 10 to 100 amino acid residues, which were found in intergenic regions previously thought to lack normal ORFs, by applying the technique described in Tomato. is.

<4. Synthesis of functional peptide>
In this example, among the identified peptides, a peptide of 41 amino acid residues named TsORF17 (MDFHDVWRSNIQDVRKLALETSKCVLMGPSLFGRVKFICFG: SEQ ID NO: 1) was analyzed. In this example, TsORF17 and partial peptides (TsORF17-1 and TsORF17-2) obtained by dividing TsORF17 into the following two regions were chemically synthesized.
TsORF17-1: MDFHDVWRSNIQDVRKLALETSK (SEQ ID NO: 3)
TsORF17-2: LETSKCVLMGPSLFGRVKFICFG (SEQ ID NO: 2)

<5. Evaluation of antibacterial activity against plant pathogenic fungi>
5-1. Plate antibacterial test In this example, antibacterial against Botrytis cinerea, Colletotrichum destructivum, Colletotrichum orbiculare, Fusarium oxysporum and Pyricularia oryzae Activity was assessed by observing spore germination. In this example, 100 μl (approximately 10 ⁴ spores) of a spore suspension of these filamentous fungi was first dispensed into a 96-well microplate.

Peptides to be tested (TsORF17-1 and TsORF17-2) were then added to each well. The peptide concentration was 1 μM, 10 μM or 100 μM. After that, it was cultured at 25° C. for 1 or 2 days.

After culturing, each well was observed under a microscope to evaluate the antibacterial activity of the tested peptides. In this example, levels 0 to 3 were defined in descending order of antibacterial activity. As a result of microscopic observation, if the hyphae extend over the entire well and no antibacterial activity is observed, it is set as "level 0", and if the hyphae extend to less than half of the well and the growth is evacuated, it is set as "level 1". , When there are ungerminated spores and some germination is inhibited, it is set as "level 2", and when all the spores are ungerminated and germination is completely inhibited, it is set as "level 3".

5-2. Botrytis cinerea leaf inoculation test In this example, a suspension containing Botrytis cinerea and a test peptide was dropped on a plant leaf, and lesions were visually observed to determine whether the test peptide was present. Antibacterial activity was evaluated.

First, spores of Botrytis cinerea were suspended in potato decoction medium (PDB) to a spore concentration of 5×10 ⁵ /ml to prepare a PDB suspension spore solution. Next, test peptides (TsORF17-2 and TsORF17 (full length)) were added to the PDB suspension spores at final concentrations of 0, 1 µM, 10 µM or 100 µM.

Next, 10 µl of the PDB suspension spore solution containing the peptide to be tested was added dropwise to the front and back of the leaf of tomato (variety name "Momotaro Fight"). In this state, the cells were incubated at 25°C for 2 days. After that, the front and back sides of the leaves were visually observed. Fig. 1 shows a photograph of the surface of a leaf to which the TsORF17-2-added PDB suspension spore solution was dropped. As shown in FIG. 1, gray mold lesions were observed even when a PDB suspension spore solution containing 100 μM TsORF17-2 was dropped. Although not shown, lesions were also observed at all concentrations when TsORF17 (full length) was used. These results suggest that TsORF17-2 and TsORF17 do not exhibit antibacterial activity against Botrytis under the conditions of this example.

5-3. Leaf inoculation test of anthracnose fungus In this example, a suspension containing Colletotrichum destructivum and a test peptide was added dropwise to plant leaves, and lesions were visually observed to determine the The antibacterial activity of peptides was evaluated.

First, spores of Colletotrichum destructivum were suspended in water to a spore concentration of 5×10 ⁵ /ml to prepare a water-suspended spore solution. Next, test peptides (TsORF17-2 and TsORF17 (full length)) were added to the water-suspended spore solution so that the final concentrations of the test peptides were 0, 1 µM, 10 µM or 100 µM.

Next, 10 µl of the water-suspended spore solution containing the test peptide was dropped on the front and back of the leaves of Chinese cabbage (variety name "Yellow Gokoro 85"). In this state, the cells were incubated at 25° C. for 4 days. After that, the front and back sides of the leaves were visually observed. FIG. 2 shows a photograph of the surface of a leaf onto which the water-suspended spore solution containing TsORF17-2 was dropped. As shown in FIG. 2, with TsORF17-2, no anthracnose lesions were observed at a concentration of 100 μM, and anthracnose lesions were reduced at a concentration of 10 μM. Although not shown, when TsORF17 (full length) was used, anthracnose lesions were not observed at concentrations of 100 μM and 10 μM. These results revealed that TsORF17-2 and TsORF17 (full length) exhibited extremely excellent antibacterial activity against cruciferous anthracnose.

5-4. Results Table 1 summarizes the results of experiments 5-1, 5-2 and 5-3 above.

As shown in Table 1, the peptide of TsORF17-2 (SEQ ID NO: 2) was isolated from Botrytis cinerea, Colletotrichum destructivum, Colletotrichum orbiculare and blast fungus ( Pyricularia oryzae) was able to inhibit the germination of a wide range of phytopathogenic filamentous fungi. Moreover, as shown in Table 1, TsORF17 (SEQ ID NO: 1) was also able to inhibit germination of plant pathogenic fungi such as Colletotrichum destructivum.

In Table 1, the full-length peptide of TsORF17 (SEQ ID NO: 1) and the partial peptide of TsORF17-2, which had no germination inhibitory effect against these plant pathogenic filamentous fungi, were similarly germinated by examining various conditions such as concentration. May exhibit inhibitory effects.

Claims (9)

A peptide consisting of the amino acid sequence: MDFHDVWRSNIQDVRKLALETSKCVLMGPSLFGRVKFICFG (SEQ ID NO: 1) or consisting of the amino acid sequence: LETSKCVLMGPSLFGRVKFICFG (SEQ ID NO: 2) and exhibiting a bactericidal or antibacterial action against plant pathogens . A nucleic acid encoding the peptide of claim 1 . A recombinant vector comprising the nucleic acid according to claim 2 . A transformant having the recombinant vector according to claim 3 . 5. The transformant according to claim 4 , which is a plant cell or plant transformed with the recombinant vector. A composition comprising the peptide of claim 1 and exhibiting bactericidal or antibacterial activity against plant pathogens. An agricultural chemical comprising the peptide according to claim 1 . 8. The agricultural chemical according to claim 7 , which is for sterilization or antibacterial use against plant pathogenic bacteria. The plant pathogen is selected from the group consisting of Botrytis cinerea, Colletotrichum destructivum, Colletotrichum orbiculare, Fusarium oxysporum and Pyricularia oryzae. 9. The pesticide according to claim 8 , which is one or more plant pathogenic filamentous fungi.

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