JP2024009531A - Novel polypeptide, novel polynucleotide and applications thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To search for a novel substance with anti-filamentous fungal activity from the secretions of Rhizoctonia solani.

SOLUTION: The present invention provides a polypeptide which includes an amino acid sequence represented by SEQ ID NO: 1, or a polypeptide with anti-filamentous fungal activity, which includes an amino acid sequence wherein one or several amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1.

SELECTED DRAWING: Figure 13

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention relates to novel polypeptides, novel polynucleotides and their uses.

Bacteria of the genus Rhizoctonia have attracted attention for a long time because they contain many plant pathogenic bacteria, such as sheath blight of rice and black blight of potatoes, and more than 100 species of Rhizoctonia have been reported so far. ing. When Rhizoctonia solani grows in bran medium, it secretes hydrolytic enzymes such as endo-β-1,3-glucanase and protease, which generally dissolve yeast cell walls. (see non-patent documents 1 to 3).

On the other hand, it has been reported that the secreted substance of Rhizoctonia solani also exhibits hyphal growth and conidiation inhibiting properties of rice-transmissible filamentous fungi (see, for example, Patent Document 1).・It is emphasized that the β-1,3-glucanase secreted from the D138 strain of P. solani contains both exo-type, endo-type, and exo-type, and exhibits growth inhibiting properties of rice-transmissible filamentous fungi. It is being Therefore, it has been suggested that Rhizoctonia solani or its culture can control plant diseases caused by microorganisms, especially filamentous fungi.

Totani K, Harumiya S, Nanjo F, et al. , Substrate affinity chromatography of β-1,3-glucanase from Basidiomycetes species, Agric. Biol. Chem. , 47(5), 1159-1162, 1982 Usui T, Oguchi M, Purification of a protease from Rhizoctonia solani lysing yeast cell walls, Agric. Biol. Chem. , 50(2), 535-537, 1986 Totsuka A, Usui T, Separation and characterization of the endo-β-(1->3)-D-glucanase from Rhizoctonia solani, Agric. Biol. Chem. , 50(3), 543-550, 1986

JP2021-20864

The purpose of the present invention is to search for novel substances having anti-fungal activity from secreted substances of Rhizoctonia solani.

Based on this background, from the secreted substances of Rhizoctonia solani, exo-type and endo-type β-1,3-glucanases, proteases and other hydrolytic enzymes known to exhibit cell membrane solubility similar to glucanases. As a result, we obtained a novel polypeptide with a molecular weight of about 10 kDa that exhibits the ability to inhibit the growth of filamentous fungi as described below. That is, the present invention includes the following embodiments.

(1) A polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or a polypeptide having an amino acid sequence in which one or several amino acids have been deleted, substituted, or added, and which has anti-mycotic activity. peptide.
(2) A polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or a polypeptide having an amino acid sequence having 70% or more sequence identity with the amino acid sequence, and having anti-filamentous activity.
(3) A polynucleotide having the 155th to 418th base sequence of the base sequence represented by SEQ ID NO: 2, or a polynucleotide having a base sequence with 70% or more sequence identity to the polynucleotide and having anti-filamentous activity. A polynucleotide encoding a peptide.
(4) An expression vector comprising the polynucleotide according to (3).
(5) A derivative or modified form of the polypeptide according to (1) or (2).
(6) A composition for controlling plant-transmissible filamentous fungi, comprising the polypeptide according to (1) or (2), or its derivative or modified form.
(7) A pesticide comprising the polypeptide according to (1) or (2), or a derivative or modified form thereof.

The polypeptides and polynucleotides of the present invention can be used as novel substances with anti-mycotic activity.

FIG. 1 is a chromatogram obtained by fractionating a secretory substance sample derived from Rhizoctonia solani by CM column chromatography. FIG. 2 shows the results of evaluating the inhibition of hyphal growth of filamentous fungi using the agar-well method for fractions F35 to F40, which were fractionated by CM column chromatography. FIG. 3 shows the results of SDS-PAGE analysis of fractions separated by CM column chromatography. FIG. 4 is a chromatogram when fractions F37 and F38, which were fractionated by CM column chromatography, were further fractionated by SP column chromatography. FIG. 5 shows the results of evaluating the inhibition of hyphal growth of filamentous fungi using the agar-well method using the fractions obtained by SP column chromatography. FIG. 6 shows the results of analyzing the fractions separated by SP column chromatography by SDS-PAGE. FIG. 7 is a chromatogram obtained when the fraction fractionated by SP column chromatography was fractionated by Capto HiRes S column chromatography. FIG. 8 shows the results of evaluating the hyphal growth inhibition of filamentous fungi using the agar-well method using the fractions eluted by Capto HiRes S column chromatography. FIG. 9 shows the results of SDS-PAGE analysis of the fractions eluted by Capto HiRes S column chromatography. Figure 10 is the cDNA and amino acid sequence of the 10 kDa anti-fungal polypeptide. FIG. 11 shows the insert and surrounding sequences of a 10 kDa anti-fungal polypeptide expression vector. FIG. 12 shows the results of SDS-PAGE analysis of recombinant proteins expressed in E. coli. FIG. 13 shows the results of measuring the filamentous fungus growth inhibiting activity of the recombinant protein expressed in E. coli using the agar-well method.

Next, each embodiment of the present invention will be described with reference to the drawings. It should be noted that each embodiment described below does not limit the claimed invention, and all of the various elements and combinations thereof described in each embodiment are the solution of the present invention. is not necessarily required.

(Novel polypeptide)
A polypeptide according to one embodiment of the present invention has the following amino acid sequence. Note that a polypeptide "having" a specific amino acid sequence, etc. includes a polypeptide "consisting of" the amino acid sequence, etc., and a polypeptide "comprising" the amino acid sequence, etc. . In addition, in this specification, in accordance with the convention for peptide markings, the left end is the N-terminus (amino terminal), and the right end is the C-terminus (carboxy terminus).

N-terminal side ETSYGNPDLVTDQGNRFKLNFGSTDGHPNACPGHYICYISKNGAQHGDVSLGNPDDFVDEGNRWRLNYGSTDGHPNACPGHYICYISK (SEQ ID NO: 1)

The polypeptide according to this embodiment may be a polypeptide having homology in which one to several amino acids are deleted, added, and/or substituted in the amino acid sequence represented by SEQ ID NO: 1. Those having filamentous fungal activity are included. In the present specification, when referring to "a polypeptide in which one to several amino acids are deleted, added, and/or substituted", the number of those amino acids is determined as long as the polypeptide has anti-mycotic activity. Although not particularly limited, the number is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1, 2, or 3. The deletion, addition, and/or substitution location may be at the end or middle of the peptide, and may be at one location or at two or more locations.

As used herein, "anti-filamentous fungi activity" may be either bacteriostatic activity or bactericidal activity against filamentous fungi. For example, it may mean any of the following: inhibition or suppression of the elongation of hyphae of filamentous fungi, inhibition or suppression of conidia formation of filamentous fungi, inhibition or suppression of formation of mycelia of filamentous fungi, and the like. As used herein, filamentous fungi are eukaryotes belonging to the phylum Fungi or Osteomycota, which include Basidiomycota, Ascomycota, Deuteromycota, Zygomycota, and Flagellate. They are microscopic microorganisms that form filamentous, branched vegetative bodies and spores, with cell walls made of chitin/glucan or cellulose glucan.

As an amino acid sequence in which one to several amino acids are deleted, added, and/or substituted in the above amino acid sequence, the above amino acid sequence and the BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) united states at least 50%, preferably 70% or more, more preferably 80%, when calculated using the National Center for Biological Information's Basic Local Alignment Search Tool) etc. (for example, using default or initial setting parameters). % or more, particularly preferably 90% or more.

The polypeptide according to this embodiment also includes various derivatives and/or modifications thereof, as long as they solve the problems of the present invention. A polypeptide derivative is a polypeptide whose amino acid sequence has been changed due to amino acid substitution, deletion, or insertion, or a polypeptide whose amino acid sequence has been changed by amino acid substitution, deletion, or insertion, or which has a chemically modified amino acid sequence such as glycosylation, glycosylation deletion, unnatural amino acid insertion, ring insertion, or methyl residue. Includes modifications, proteolytic fragments as well as deletion fragments. These polypeptide derivatives can be naturally occurring or non-naturally occurring. Non-naturally occurring derivatives can be produced using mutagenesis techniques known in the art. Polypeptide derivatives include polypeptides that have one or more residues chemically derivatized by reaction of functional side groups. Similarly, "derivatives" include those peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be used in place of proline, 5-hydroxylysine can be used in place of lysine, 3-methylhistidine can be used in place of histidine, and homoserine can be used in place of serine. can be used in place of lysine, and ornithine can be used in place of lysine.

The term "modified product" refers to the polypeptide of this embodiment that has been chemically or biologically modified. Chemical modifications include bonding of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked sugar chains, and the like. Examples of chemical moieties included in the chemically modified product include water-soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, and polyvinyl alcohol. Biological modifications include post-translational modifications (e.g. N- or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation) These include those in which a methionine residue is added to the N-terminus by expression using a prokaryotic host cell, and fusions in which other peptides such as tags are added by genetic recombination. Furthermore, enzyme labels, fluorescent labels, and affinity labels are also included in the meaning of such modifications.

Production of polypeptides according to this embodiment may be accomplished by heterologous expression of suitable nucleic acid constructs in various host organisms and host cells. The necessary genetic engineering methods are described in Sambrook and Russell 2001 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NJ. Prokaryotic systems (eg, E. coli) or eukaryotic systems (eg, insect cells, plant cells, or mammalian cells) can be used.

Alternatively, the polypeptide according to this embodiment can be produced by a chemical peptide synthesis method. For example, B. This may involve the use of known solid phase methods according to Merrifield. Synthesis can be performed with the aid of an automatic peptide synthesizer or manually using Fmoc or Boc protecting group strategies. Polypeptides can be synthesized by later joining smaller fragments together.

(New polynucleotide)
A polynucleotide according to another embodiment of the present invention has base sequences 155 to 418 (underlined) in the following base sequence.
５'－ＡＴＣＣＡＡＣＡＧＡＴＣＴＣＧＴＣＴＣＡＡＧＣＴＣＡＣＴＴＴＣＣＡＡＧＣＡＣＡＣＡＣＣＡＡＣＴＣＡＧＣＣＡＴＧＴＴＴＴＣＣＴＣＴＧＣＴＣＡＴＣＴＣＧＣＴＴＴＣＴＴＴＧＣＴＧＣＴＧＣＴＧＣＴＧＣＣＧＣＴＣＴＴＧＴＴＧＣＴＧＣＡＡＧＣＣＣＴＧＴＣＡＣＴＧＡＧＣＴＣＧＡＡＧＧＡＣＡＧＡＣＴＣＴＴＧＣＣＡＡＧＣＧＣ ＧＡＧＡＣＧＴＣＡＴＡＣＧＧＣＡＡＴＣＣＣＧＡＣＴＴＧＧＴＡＡＣＴＧＡＣＣＡＧＧＧＣＡＡＴＣＧＣＴＴＣＡＡＧＣＴＣＡＡＣＴＴＴＧＧＣＡＧＣＡＣＣＧＡＣＧＧＣＣＡＣＣＣＧＡＡＣＧＣＧＴＧＴＣＣＣＧＧＡＣＡＣＴＡＣＡＴＣＴＧＣＴＡＣＡＴＣＴＣＡＡＡＧＡＡＣＧＧＡＧＣＣＣＡＡＣＡＴＧＧＣＧＡＣＧＴＣＴＣＴＣＴＣＧＧＣＡＡＴＣＣＣＧＡＣＧＡＣＴＴＴＧＴＡＧＡＴＧＡＧＧＧＣＡＡＣＣＧＴＴＧＧＣＧＣＣＴＧＡＡＣＴＡＴＧＧＴＡＧＣＡＣＣＧＡＴＧＧＡＣＡＣＣＣＧＡＡＣＧＣＣＴＧＣＣＣＴＧＧＡＣＡＣＴＡＣＡＴＣＴＧＣＴＡＴＡＴＣＡＧＣＡＡＧ ＴＧＡＡＣＴＴＡＴＣＡＡＣＴＴＡＡＣＴＣＧＴＣＡＴＴＴＣＣＣＧＣＡＧＴＴＴＣＣＴＴＴＡＴＧＴＴＴＧＴＡＴＴＡＣＴＴＣＴＣＡＧＴＴＡＡＡＴＣＡＡＣＣＡＴＡＧＧＡＡＡＴＡＴＡＧＣＴＡＴＣＴＴＴＡＡＣＡＣＣＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡＡ－３'（配列番号２）

In one embodiment, the polynucleotide having the 155th to 418th base sequence of the base sequence represented by SEQ ID NO: 2 has a predetermined sequence identity with the 155th to 418th base sequence of the base sequence represented by SEQ ID NO: 2. It may be a polynucleotide having a nucleotide sequence with a specific character. The degree of base sequence identity is about 70% or more, preferably about 90% or more, more preferably about 95% or more, even more preferably about 96% or more, most preferably about 97% or more, about 98% or more. or about 99% or more. Base sequence identity can be determined by a method known per se. For example, base sequence identity (%) can be determined by the same method as the amino acid sequence identity (%) described above.

In another embodiment, the polynucleotide has one or more modifications selected from substitution, addition, deletion, and insertion of one or more nucleotides in the 155th to 418th base sequence of the base sequence represented by SEQ ID NO: 2. It may be a modified base sequence. The number of modified nucleotides is not particularly limited as long as it is 1 or more, but for example, 1 to about 100, preferably 1 to about 70, more preferably 1 to about 50, even more preferably 1 to about 30. , most preferably 1 to about 20, 1 to about 10, or 1 to about 5 (eg, 1 or 2).

In yet another embodiment, the polynucleotide may be a polynucleotide that can hybridize to the 155th to 418th base sequence of the base sequence represented by SEQ ID NO: 2 under high stringency conditions. Conditions for hybridization under high stringency conditions can be set with reference to previously reported conditions (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6 ( (1999)). For example, conditions for hybridization under high stringency conditions include hybridization at 6×SSC (sodium chloride/sodium citrate)/45°C, followed by 0.2×SSC/0.1% SDS/50-65 One or more washes at <RTIgt;C</RTI> are included.

A polynucleotide of this embodiment may encode a polypeptide of this embodiment. Therefore, the polynucleotide of this embodiment can be a polynucleotide such that the polypeptide encoded thereby can exhibit a similar function as the polypeptide of this embodiment.

(expression vector)
The expression vector according to one embodiment of the present invention may contain a polynucleotide encoding a polypeptide of interest to be expressed or a polynucleotide of interest to be expressed, and a promoter operably linked to the polynucleotide. "A promoter is operably linked to a polynucleotide" means that the promoter is operably linked to the polynucleotide in such a way that the promoter enables expression of the polynucleotide itself or a polypeptide encoded by the polynucleotide under its control. It means that it is linked to a polynucleotide encoding.

The backbone of the expression vector of this embodiment is not particularly limited as long as it can produce the desired substance in a predetermined cell, and examples thereof include plasmid vectors and viral vectors. When the expression vector is used as a medicine, vectors suitable for administration to mammals include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, Sindbis virus, Sendai virus, etc. Examples include viral vectors.

When using prokaryotic cells as host cells, expression vectors that can utilize prokaryotic cells as host cells can be used. Such expression vectors may contain elements such as, for example, a promoter-operator region, an initiation codon, a polynucleotide encoding the polypeptide of this embodiment or a partial peptide thereof, a stop codon, a terminator region, and an origin of replication. A promoter-operator region for expressing a polypeptide of the invention in bacteria is one that includes a promoter, an operator, and a Shine-Dalgarno (SD) sequence. As these elements, those known per se can be used.

Furthermore, when using eukaryotic cells as host cells, expression vectors that can utilize eukaryotic cells as host cells can be used. In this case, the promoter used is not particularly limited as long as it can function in eukaryotes such as mammals. When the purpose is to express a polypeptide, such promoters include, for example, viral promoters such as SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter, and β - Examples include mammalian constituent protein gene promoters such as actin gene promoter, PGK gene promoter, and transferrin gene promoter. If the purpose is to express a polynucleotide, the promoter can be a polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter).

The expression vectors of the invention further contain sites for transcription initiation and termination, and ribosome binding sites that may be required for translation in the transcribed region, origins of replication, and selectable marker genes (e.g., ampicillin, tetracycline, kanamycin, spectinomycin). , erythromycin, chloramphenicol) and the like. The expression vector of the present invention can be produced by a method known per se (see, for example, Molecular Cloning, etc., listed above).

(Composition for controlling plant-transmissible filamentous fungi)
The composition for controlling plant-transmissible filamentous fungi of this embodiment (hereinafter referred to as "composition of this embodiment") is characterized by containing the above-mentioned polypeptide, its derivative, or its modified form. The composition of this embodiment can be manufactured according to a conventional method. That is, the composition of this embodiment can contain other active ingredients as long as it contains at least one of the above polypeptides, derivatives thereof, or modified forms thereof as active ingredients. Furthermore, carriers can be mixed and used, and if necessary, formulation auxiliaries such as surfactants, wetting agents, fixing agents, thickeners, antibacterial and fungicidal agents, colorants, and stabilizers may be added. can be added and formulated into, for example, granules, wettable powders, flowables, wettable powders, powders, emulsions, etc. according to conventional methods.

The content of the active ingredient in the composition of the present embodiment is usually in the range of 0.005 to 99% by weight, preferably in the range of 0.1 to 90%, more preferably 0. .3 to 80%. The content of the active ingredient in the composition of the present embodiment varies depending on the formulation and is selected as appropriate, but is usually 0.01 to 30% by weight for powders and 0.1 to 30% by weight for wettable powders. 80% by weight for granules, 0.5 to 25% by weight for emulsions, 2 to 50% by weight for emulsions, 1 to 50% by weight for flowable formulations, and 1 to 50% for dry flowable formulations. It is 80% by weight.

The composition of this embodiment is effective, for example, against plant diseases originating from various filamentous fungi, and is preferably effective for controlling plant-transmissible filamentous fungi. Examples of specific diseases to be controlled by the composition of the present embodiment include, for example, rice blast (Pyricularia oryzae), sheath blight (Thanatephorus cucumeris), sesame leaf blight (Cochliobolus miyabeanus), and rice blight (Cochliobolus miyabeanus). Disease (Gibberella fujikuroi); Powdery mildew (Erysiphe graminis), rust (Puccinia striiformis), spotted leaf disease (Pyrenophora graminea), net spot disease (Pyrenophora maize head blight (Fusarium graminearum, etc.); Seedling damping-off (Fusarium avenaceum), sesame leaf blight (Cochliobolus heterostrophus), smut (Ustilago maydis); pear black spot (Alternaria alternata), scab (Venturi) a nashicola), Gymnosporangium haraeanum; peach Cladosporium carpophilum, Phomopsis sp., Phytophthora sp.; Glomerella cingulata, Monilinia ku sanoi), Monilinia fructicola; persimmons Anthracnose (Gloeosporium kaki), defoliation (Cercospora kaki), powdery mildew (Phyllactinia kakikora); citrus black spot (Diaporthe citri), green mold (Penicillium digitat) um), blue mold (Penicillium italicum); tomatoes, cucumbers, beans , Botrytis cinerea on strawberries, potatoes, cabbages, eggplants, lettuce, etc.; Sclerotinia sclerotiorum on tomatoes, cucumbers, beans, strawberries, potatoes, rapeseed, cabbage, eggplants, lettuce, etc.; Sclerotinia sclerotiorum on tomatoes, cucumbers, etc. , beans, radish, watermelon, eggplant, rapeseed, green pepper, spinach, sugar beet, and other vegetable seedling damping-off (Rhizoctonia spp.); cucurbit downy mildew (Pseudoperonospora cubensis), cucumber powdery mildew (Sphaerotheca cucurbita) e); Tomato rot (Alternaria solani), leaf mold (Cladosporium fulvam), tomato powdery mildew (Oidium neolycopersici); cabbage stock rot (Rhizoctonia solani), yellowing disease (Fusarium oxy) Chinese cabbage butt rot (Rhizoctonia solani) , Verticillium dahlie; Puccinia allii, Alternaria porri; Cercospora kikuchii, Elsinoe glycinnes, black spot Diseases (Diaporthe phaseolum) etc. These include, but are not limited to:

(Pesticides)
As described above, the agricultural chemical of this embodiment is characterized by containing the above-mentioned polypeptide, its derivative, or its modified form. According to the agricultural chemical of this embodiment, for example, it is possible to control plant infectious diseases caused by microorganisms. For the agricultural chemical of the present invention, the explanation of the composition for controlling plant-transmissible filamentous fungi can be referred to.

The dosage form of the agricultural chemical of this embodiment is not particularly limited, and examples thereof include solid dosage forms such as powders, fine granules, and granules, liquid preparations, emulsions, and the like. The agricultural chemicals of this embodiment may include, for example, known agricultural chemicals. The known pesticide may be, for example, a biological pesticide or a chemical pesticide. The biological pesticide is, for example, a pesticide containing microorganisms (eg, bacteria, fungi) having activities such as antibacterial activity, insecticidal activity, fungicidal activity, herbicidal activity, plant growth regulating activity, and insect repellent activity. Further, examples of the chemical pesticides include antibacterial substances, insecticidal substances, bactericidal substances, herbicidal substances, plant growth regulating substances, insect repellent substances, and the like.

The agricultural chemicals can be used for, but are not particularly limited to, examples thereof include plants, soil for cultivating the plants, and media such as water. Examples of the plants include gramineous plants such as rice, and paddy rice is preferred. When the pesticide is used on the plant, the position in the plant where the pesticide is brought into contact is not particularly limited, and may be the entire plant or a part of the plant. The part of the individual plant may be any organ, tissue, cell, vegetative propagation body, etc., for example. Examples of the organs include petals, corollas, flowers, leaves, seeds, fruits, stems, roots, and the like. As a specific example, the objects to be used include, for example, rice seeds (seeds), seedlings, and the like. The growth state of the plant when using the above-mentioned agricultural chemical is not particularly limited, and for example, it may be a seed, a seedling, or a grown body.

The amount and frequency of use of the agricultural chemical are not particularly limited, and can be set as appropriate, for example, depending on the target of use. The agricultural chemical of this embodiment can be used, for example, according to a general usage method. The method of use includes, for example, a method of directly spraying the pesticide by hand, a backpack-type granulator, a pipe granulator, an aerial granulator, a power granulator, a granulator for seedling boxes, and a multi-mouth hose granulator. Examples include a method of scattering the grains using a granulator such as a granulator attached to a rice transplanter or rice transplanter. Furthermore, when the agricultural chemical of the present invention is a liquid agent, the agricultural chemical can be applied using a sprayer such as a backpack sprayer, a power sprayer, a sprinkler, a type of sprayer mounted on a tractor, or a multi-nosed hose sprayer. It can be sprayed by

The place where the agrochemical of the present invention can be used is not particularly limited, and can be used, for example, on agricultural land such as paddy fields, dry fields, seedling boxes, fields, orchards, mulberry fields, greenhouses, and open fields.

The case where the agricultural chemical of the present invention is used for rice will be explained by giving an example. When using the agrochemical of the present invention for rice, the agrochemical of the present invention can be used, for example, at the time of sowing, seedling raising, rice transplanting, and the like. The agricultural chemical of the present invention may be used, for example, in an undiluted state or in a diluted state. Can be dispersed or dispersed using a sprinkler, etc. Furthermore, when the agricultural chemical of the present invention is used to disinfect the rice seeds, the agricultural chemicals can be used, for example, as a coating agent for the rice seeds. For example, the agricultural chemical of the present invention may be further used by being dissolved in soaking water when soaking the rice seeds, or may be used by being dissolved in hot water when disinfecting the rice seeds. Alternatively, the water may be sprayed during germination of the rice seeds.

When used in the rice field, the amount and frequency of use of the agricultural chemical of the present invention are not particularly limited, and can be appropriately determined depending on the size of the rice field and the usage method.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples in any way.

[Example 1] Purification of a novel anti-filamentous fungal polypeptide (preparation of secreted substance sample and fractionation by acid precipitation and CM column chromatography)
The secreted substance sample used for this purification was a dried powder of secreted enzymes of Rhizoctonia solani strain D138 (Fuji Film Wako Pure Chemical Industries, Ltd. and K.I. Kasei Co., Ltd.) prepared by the method described in Patent Document 1.

2 g of the above dry powder was weighed and dissolved in 20 mL of 50 mM acetate buffer (pH 4.8) (hereinafter referred to as "buffer A") to obtain a dry powder solution. The resulting acid precipitate was removed by centrifugation at 8000 x g for 15 minutes at 4°C, and the collected supernatant was transferred to a cellulose tube 27/32 (Edia). and dialyzed against buffer A for 18 hours. The solution after dialysis was centrifuged again at 8,000 x g for 15 minutes, and the resulting supernatant was filtered through a 0.2 μm filter (DISMIC-25CS, Advantech), used as an acid precipitation supernatant solution, and subjected to column chromatography. provided.

The protein was quantified using the Quick Start Protein Assay Kit (Bio-Rad Laboratories) by the Bradford method using bovine serum albumin as a standard. The total protein amounts of the above dry powder solution and acid precipitation supernatant solution were 554 mg and 375 mg, respectively.

Column chromatography was performed using an AKTApurifier 100 chromatography system (Cytiva) in a chromatography chamber set at 4°C. The acid precipitation supernatant solution was applied to a HiTrap CM FF column (volume 5 mL, average particle size 90 μm, Cytiva), which is a weak cation exchange column. The column equilibrated with buffer A at a flow rate of 2.5 mL/min for 20 minutes was once removed from the apparatus, and 35 mL of acid precipitation supernatant solution was manually introduced using a 30 mL capacity syringe over 2.4 minutes. . The column was reattached to the device and washed with buffer A at a flow rate of 2.5 mL/min for 50 minutes, followed by a linear concentration gradient of 0 to 250 mM sodium chloride for 80 minutes (200 mL) to separate and elute the protein. Ta. Thereafter, the sodium chloride concentration was quickly raised to 1M, and the column was washed at a flow rate of 2.5 mL/min for 28 minutes, and then buffer A was pumped at a flow rate of 2.5 mL/min for 20 minutes. The wavelength of the detector was 280 nm, and the elution solution was collected in 5 mL portions (2 minutes/volume) using an automatic fraction collector. The results are shown in Figure 1.

Since the elution chromatogram shown in FIG. 1 was obtained, the antifungal activity of the column-unadsorbed fraction and fraction numbers F30 to F49 was investigated for inhibition of hyphal growth of filamentous fungi and inhibition of yeast growth.

(Evaluation of antifungal activity 1: Evaluation of hyphal growth inhibition of filamentous fungi)
The inhibition of hyphal growth of filamentous fungi was evaluated using the agar-well method. Fusarium fujikuroi strain MAFF235949, which was provided by the National Institute of Agricultural and Biological Resources, was used as the indicator bacteria in this method, and a potato dextrose agar medium (hereinafter referred to as "PDA") was used as the medium.

A spore preservation solution of MAFF235949 strain was prepared in advance according to the following procedure. The inoculum of the PDA slant medium was inoculated onto the PDA flat medium and cultured at 28° C. for approximately two weeks. A part of the grown mycelium was smeared onto a slide glass, and the presence of spores was confirmed by phase contrast observation using an Olympus BX43 system microscope (Evident). After that, 4 mL of 0.25% (v /v) Tween 20 was added and suspended using a spreader, and then the mycelia were removed by filtration through pharmacopoeial sterilized gauze. After confirming by phase contrast observation that the collected filtrate contains spores, the filtrate was centrifuged at 8000 x g for 15 minutes at 4°C, the supernatant was removed with a Pasteur pipette, and immediately 8 mL of sterile pure Water was added to suspend and wash the precipitated spores. After repeating this washing operation twice, the supernatant was centrifuged again at 8,000×g and 4° C. for 15 minutes, and the supernatant was removed with a Pasteur pipette, and 1.5 mL of sterile pure water was added to suspend the spores. Furthermore, 0.5 mL of sterilized 80% glycerol solution was added and mixed, and the mixture was dispensed and stored frozen at -80°C.

A portion of the prepared spore suspension was used to determine the spore concentration as follows. The solution was serially diluted from 1/10 to 1/10,000 times with sterile water, and 0.1 mL of each diluted solution was applied and seeded onto PDA. The number of spores that could germinate was calculated from the number of colonies produced after culturing at 28°C for 4 days, and was approximately 5 x 10 ⁷ per mL. One of the cryopreserved spore suspensions was thawed, and a diluted solution was prepared using a 20% glycerol solution to a concentration of approximately 5 x 10 ⁵ spores/mL. This was divided into portions, frozen and stored again at -80°C, and used in the following anti-mycotic activity evaluation test.

The medium used in the agar-well method is prepared by dispensing 20 mL of sterilized PDA into a sterile petri dish with a diameter of 9 cm, solidifying it, and then using a sterile glass tube with an outer diameter of about 7 mm to place the well end 2 cm from the center of the petri dish. Holes were drilled at four locations at distances of (hereinafter referred to as "PDA assay plate") and stored at 4°C until use. The dry powder solution, acid precipitation supernatant solution, and elution fraction obtained by CM column chromatography were serially diluted 2 to 16 times with buffer A, and 80 μL each was added to the wells of a PDA assay plate. As a control without anti-fungal activity, 80 μL of buffer A alone was added to the wells. After adding each sample to the well, 10 μL of a stock spore suspension of MAFF235949 strain at approximately 5×10 ⁵ cells/mL was dropwise inoculated in the center of the PDA assay plate. After culturing at 30°C for 4 days, the culture temperature was changed to 15°C and cultured for an additional 5 days until the mycelia reached within 0.5 cm of the edge of the control well. The presence or absence of the hyphal growth inhibiting effect of each sample was determined by visually comparing the distance between the well edge and the grown hyphae with that of the control, and the strength of the inhibitory effect was evaluated by the maximum dilution ratio of the test solution that showed the inhibitory effect.

The results are shown in Table 1 and FIG. 2. In Table 1, the dry powder solution (I) and the acid-precipitated supernatant (II) showed strong anti-fungal activity even at the highest dilution rate of 16 times (126 μg and 54 μg/well of protein, respectively). confirmed. After fractionation by CM column chromatography, a strong mycelial growth inhibitory effect was observed in fraction number F2, which is a fraction not adsorbed to the column, and fraction numbers F35 to F38 (fraction eluted with 50 to 69 mM sodium chloride) (Fig. 2), anti-filamentous activity was confirmed up to the maximum dilution rate of 16 times investigated (Table 1).

[Legend, etc.]
1) +: Activity, ±: Weak activity, -: No activity 2) Fraction numbers I and II indicate the dry powder solution and acid precipitation supernatant solution, respectively, and the other numbers are CM column chromatography. 3) Well position: One of the wells on the PDA assay plate was fixed as A, clockwise B, C, D.

(Evaluation of antifungal activity 2: Yeast growth inhibition)
The inhibitory effect on yeast growth was evaluated by a microbroth dilution method using a 96-well microtiter plate. Saccharomyces cerevisiae NBRC10074 strain, which was provided by the National Institute of Technology and Product Evaluation, was used as the indicator bacteria, and potato dextrose liquid medium (hereinafter referred to as "PDB") was used as the medium.

Inoculate 20 μL of NBRC10074 strain prepared in advance to a concentration of 2.4×10 ⁷ cfu/mL and cryopreserved at -80°C into 10 mL of PDB, and culture with shaking at 120 rpm at 30°C for 18 hours. A fresh yeast culture of 2.4×10 ⁷ cfu/mL was obtained. This was further diluted 100 times with 2×PDB to obtain a yeast suspension of 2.4×10 ⁵ cfu/mL. After dispensing 100 μL of buffer A into all wells of a 96-well microtiter plate (8 rows and 12 columns), add dry powder solution, acid precipitation supernatant solution, or CM column chromatography elution to each well in the first column. The fraction solution (stock solution) was dispensed in 100 μL portions (the dilution ratio was 2 times). Next, using an 8-channel multipipette, 100 μL each was taken from the wells in the first row and mixed into the second row (dilution ratio: 4 times). Furthermore, 100 μL each was taken from the wells in the second row and mixed into the third row (dilution ratio: 8 times). By repeating the same sorting and mixing operations up to the 11th column, sample dilution solutions with a dilution factor of 2 to 2048 times were prepared (the 12th column was used as a negative control containing only buffer A). Furthermore, 100 μL (2.4×10 ⁴ cfu) of the above yeast suspension was added to all wells. Through this operation, the dilution ratio of the sample solution used for activity evaluation was 4 to 4096 times. The microtiter plate was placed in a plastic bag to prevent water evaporation and cultured at 28° C. for 18 hours. The strength of yeast growth inhibition was determined by comparing the turbidity of each well with the control, and was expressed as the maximum dilution ratio of the test solution at which growth inhibition could be visually confirmed. The results are shown in Table 2 below.

As shown in Table 2, strong yeast growth inhibitory activity was confirmed in the dry powder solution and the acid precipitation supernatant solution up to a dilution rate of 256 times. Regarding the CM column chromatography fractionation solution, strong growth inhibition was observed in fraction numbers F2 and F3, which are column non-adsorbed fractions, up to 128-fold and 64-fold dilution, respectively (Table 2). On the other hand, in the elution fractions with fraction numbers F30 to F41, no clear yeast growth inhibition activity was observed even at a dilution rate of 4 times (Table 2). A different trend was observed from the results obtained in the growth inhibition evaluation (evaluation of growth inhibition), in which anti-filamentous fungal activity was detected in both fraction numbers F2 and F3 and F35 to F38.

[Usage Guide]
Judgment result +: Proliferation, ±: Weak proliferation, -: No proliferation

From the results of the above two types of antifungal activity evaluation, it is clear that there are at least two types of antifungal proteins in the secreted material sample of Rhizoctonia solani strain D138, and one of them is present in the CM column that inhibits both filamentous fungi and yeast. It has become clear that one has a non-adsorbing property (under the above experimental conditions), while the other is adsorbed and separated by the CM column, showing strong inhibitory activity only against filamentous fungi. Thereafter, further purification was carried out targeting the latter active fractions (fraction numbers F35 to F38), which have the characteristic property of acting only on filamentous fungi. The total protein amount of fraction numbers F35 to F38 was 65.3 mg as determined by the Bradford method.

(Confirmation of protein molecular composition of CM column chromatography elution fraction)
The protein composition and molecular weight of CM column chromatography fractions F35 to F38 and surrounding fractions, which have inhibitory activity only against filamentous fungi, were confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE was performed according to the commonly used method of Laemmli, and a Quick-CBB kit (Fuji Film Wako Pure Chemical Industries, Ltd.) was used for protein staining. Furthermore, BlueStar Prestained Protein Ladder (Nippon Genetics), which is a dye-bound protein marker, was used as a molecular weight marker. The results are shown in FIG. Fraction numbers F35 to F38 contained many proteins with a wide range of molecular weights, and no significant differences were observed in the migration patterns among the fractions.

(Fractionation by SP column chromatography)
The solutions of fraction numbers F37 and F38 collected by CM column chromatography were desalted and concentrated using an Amicon Ultra centrifugal ultrafiltration filter (Merck Millipore), and then filtered using a strong cation exchange column, HiTrap SP HP. It was applied to a column (capacity 1 mL, average particle diameter 34 μm, Cytiva). The entire sample (2 mL) was injected into the column that had been equilibrated by pumping buffer A at a flow rate of 1 mL/min for 10 minutes using the sample loop, and then buffer A was pumped for 6 minutes using the sample loop as a flow path. . After washing the column with buffer A for 18 minutes, a linear concentration gradient of 0 to 250 mM sodium chloride was applied for 40 minutes (40 mL) to separate and elute the protein. Thereafter, the sodium chloride concentration was quickly increased to 1M and the column was washed for 18 minutes at a flow rate of 1 mL/min, and then buffer A was pumped at a flow rate of 1 mL/min for 10 minutes. The wavelength of the detector was 280 nm, and the elution solution was collected using an automatic fraction collector. After introducing the sample, the washing solution (column non-adsorbed fraction) was collected in 2 mL portions (2 minutes/unit), and after the start of the linear salt concentration gradient, it was collected in 1 mL portions ( 1 minute/piece).

The results are shown in FIG. Since the elution chromatogram shown in Figure 4 was obtained, the following procedure was performed for fraction numbers F1 to F3, which are column non-adsorbed fractions, and fraction numbers F18 to F29 (38 to 106 mM sodium chloride elution fraction). The SP column chromatography elution fraction was subjected to evaluation of anti-filamentary fungus activity.

(Evaluation of anti-filamentous fungus activity of SP column chromatography elution fraction)
The antifungal activity was evaluated according to the method described in "Evaluation of antifungal activity 1: Evaluation of hyphal growth inhibition of filamentous fungi" above.

The results are shown in FIG. As shown in Figure 5, in fraction numbers F20 to F23 (50 to 69 mM sodium chloride elution fraction), the mycelial growth inhibitory effect on Fusarium fujikuroi MAFF235949 strain was observed, and in fraction numbers F20 to F22, the maximum Anti-filamentous activity was confirmed up to a dilution of 8 times for fraction number F23, and up to a 4 times dilution for fraction number F23 (Table 3). The total protein amount of fraction numbers F20 to F23 collected as active fractions was 2.86 mg as determined by the Bradford method.

[Usage Guide]
1) +: Activity, ±: Weak activity, -: No activity 2) Well position: One of the wells on the PDA assay plate was fixed as A, and clockwise as B, C, and D.

(Confirmation of protein molecular composition of SP column chromatography elution fraction)
The protein molecular composition was confirmed according to the method described in "Confirmation of the protein molecular composition of the CM column chromatography elution fraction" above. As a result of SDS-PAGE analysis (Figure 6), among the active fractions with fraction numbers F20 to F23, the electrophoretic patterns of fraction numbers F20 to F22 are similar, and molecular weight markers (Figure 6, lanes (denoted as "M"), it was suggested that proteins with a lower molecular weight than about 35 kDa exhibited anti-filamentous fungal activity.

(Fractionation by Capto HiRes S column chromatography)
The solutions with fraction numbers F20 to F23 collected by SP column chromatography were desalted and concentrated using an Amicon Ultra centrifugal ultrafiltration filter (Merck Millipore), and then filtered using a strong cation exchange column with high resolution. It was applied to a certain Capto HiRes S 5/50 column (capacity 1 mL, average particle size 9 μm, Cytiva).

Using the sample loop, the entire sample volume (2 mL) was injected into the column that had been equilibrated by pumping Buffer A at a flow rate of 0.5 mL/min for 20 minutes, and then Buffer A was pumped for 12 minutes using the sample loop as a flow path. It liquefied. After washing the column with buffer A for 40 minutes, a linear concentration gradient of 0 to 150 mM sodium chloride was applied for 80 minutes (40 mL) to separate and elute the protein. Thereafter, the sodium chloride concentration was quickly increased to 1M and the column was washed at a flow rate of 0.5 mL/min for 20 minutes, and then buffer A was pumped at a flow rate of 0.5 mL/min for 10 minutes. The wavelength of the detector was 280 nm, and the elution solution was collected using an automatic fraction collector. After introducing the sample, the washing solution (column non-adsorbed fraction) was collected in 2 mL portions (4 minutes/volume), and after the start of the linear salt concentration gradient, it was collected in 1 mL portions ( 2 minutes/piece).

As a result, the elution chromatogram shown in Figure 7 was obtained, so for fraction numbers F17 to F31 (15 to 68mM sodium chloride elution fraction), the following "Capto HiRes S column chromatography elution fraction Filamentous fungi activity evaluation.

(Evaluation of anti-filamentous fungus activity of Capto HiRes S column chromatography elution fraction)
The antifungal activity was evaluated according to the method described in "Evaluation of antifungal activity 1: Evaluation of hyphal growth inhibition of filamentous fungi" above.

As a result, in fraction numbers F19 to F21 (23 to 30 mM sodium chloride elution fraction), an inhibitory effect on hyphal growth against Fusarium fujikuroi MAFF235949 strain was observed (Figure 8), and at the maximum dilution rate of 4 times and protein content Even at 1.1 μg/well, clear anti-filamentous activity was confirmed (Table 4). The total protein amount of fraction numbers F19 to F21 collected as active fractions was 0.187 mg as determined by the Bradford method. This amount of protein was the final yield of the anti-mycotic active polypeptide after the series of purifications up to this point, and the recovery rate in terms of protein amount was 0.034%.

[Usage Guide]
1) +: Activity, -: No activity 2) Well position: One of the wells on the PDA assay plate was fixed as A, and clockwise as B, C, and D.

(Confirmation of protein molecular composition of Capto HiRes S column chromatography elution fraction)
The protein molecular composition was confirmed according to the method described in "Confirmation of the protein molecular composition of the CM column chromatography elution fraction" above. As a result of SDS-PAGE analysis, a single protein band was detected in fraction numbers F19 to F21, and referring to the molecular weight marker (labeled lane "M" in Figure 9), a polypeptide of around 10 kDa was detected. was purified as an anti-mycotic active substance (Figure 9).

[Example 2] Analysis of amino acid sequence information of 10 kDa anti-filamentous fungal polypeptide (analysis of N-terminal amino acid sequence of purified protein and homology search)
The N-terminal amino acid sequence of the anti-filamentous fungal protein obtained in Example 1 with a molecular weight of approximately 10 kDa was analyzed using a purified preparation (Capto HiRes S column chromatography fraction number F20 was used).

A solution of fraction number F20 with an amount of 8.1 μg of protein was subjected to SDS-PAGE, and the protein was extracted from the polyacrylamide gel after electrophoresis using a tank-type blotting device (Mini Transblot Cell, Bio-Rad Laboratories). was transferred to a PVDF membrane (Immobilon-PSQ membrane, 0.2 μm, Merck & Co.). After the transfer, the PVDF membrane was stained in a 0.1% Coomassie Brilliant Blue R250 solution dissolved in 50% methanol for 2 minutes with gentle shaking, then decolorized with 50% methanol, and further washed with ultrapure water. Excess dye on the membrane was removed. Thereafter, a protein band with a molecular weight of approximately 10 kDa was cut out from the PVDF membrane dried for 18 hours and subjected to N-terminal amino acid sequence analysis.

N-terminal amino acid sequence analysis was performed by automated Edman degradation using a gas phase sequencer (491cLc, Applied Biosystems). The handling and various settings of the equipment were performed according to the procedures specified by the manufacturer. As a result, the N-terminal amino acid sequence of 13 residues shown below was obtained.
N-terminal side ETSYGNPDLVTDQ (SEQ ID NO: 3)

As a result of an amino acid sequence homology search for this 13-residue partial amino acid sequence using the BLAST search system provided by the National Center for Biotechnology Information (NCBI), the following Hypothetical protein RHS04_00525 [Rhizoctonia solani] was found to have 100% homology. ], Accession number: KAF8685646.1 was presented.

N-terminal side MKRHPKKYQTETTDAVAASRLKLTFQAHTNSAMFSSAHLAFFAAAAALVAASPVTELEGQTLAKR ETSYGNPDLVTDQ GNRFKLNFGSTDGHPNACPGHYICYISKNGAQHGDVSL GNPDDFVDEGNRWATPELW (SEQ ID NO: 4)

The underline in the above amino acid sequence indicates a sequence with 100% homology to SEQ ID NO: 3. The results of this homology search revealed that this polypeptide exhibits high homology with proteins whose biological functions are unknown, strongly suggesting that it is a novel anti-fungal polypeptide.

(Estimation of 10kDa anti-filamentous fungus polypeptide sequence based on whole genome information of Rhizoctonia solani strain D138)
In addition to the analysis based on the information obtained from the amino acid sequence analysis of the purified protein described above, we determined the entire genome sequence of Rhizoctonia solani strain D138, which is the strain producing this polypeptide, and attempted an analysis based on that information. .

Genomic DNA used for whole genome sequence analysis was prepared as follows. For Rhizoctonia solani strain D138, a part of mycelium was scraped off from a seed strain that had been subcultured on a PDA slanted medium, and this was inoculated onto a PDA slanted medium and cultured at 30° C. for about 8 days. Next, 4 mL of sterile pure water was added to this PDA flat medium and suspended using a spreader, and a portion was collected into a 1.5 mL microtube. Subsequently, this microtube was centrifuged at 6,000×g, 4° C., for 5 minutes, and the supernatant was removed. 5 μL of sterile pure water was again added to the remaining mycelial precipitate, and the entire amount was recovered into a tube containing stainless steel beads for crushing. Lysis Solution F (Nippon Gene Co., Ltd.) was added to this, and the mixture was ground for 2 minutes at 1500 rpm using Shake Master Neo (BMS Co., Ltd.). The crushed sample was incubated at 65° C. for 10 minutes, and then centrifuged at 12,000×g for 2 minutes to collect the supernatant. DNA was purified from the collected supernatant using the MPure-12 system and MPure Bacterial DNA Extraction Kit (both manufactured by MP Bio).

For library preparation, the concentration of the genomic DNA solution prepared above was first measured using Synergy LX (Bio Tek) and QuantiFluor dsDNA System (Promega). Next, a library was prepared using MGIEasy FS DNA Library Prep Set (MGI) according to the manufacturer's instructions. The reaction time for enzymatic cleavage was 4 minutes. In addition, an adapter from the MGIEasy DNA Adapters-96 (Plate) Kit (MGI) was used at that time. Quantification of the library was performed using Qubit 3.0 Fluorometer and dsDNA HS Assay Kit (both from Thermo Fisher Scientific). The quality of the library was checked using Fragment Analyzer and dsDNA 915 Reagent Kit (both from Advanced Analytical Technologies). Circularized DNA was prepared using the prepared library and MGIEasy Circularization Kit (MGI) according to the manufacturer's instructions. DNA Nanoball (hereinafter referred to as "DNB") was prepared using DNBSEQ-G400RS High-throughput Sequencing Set (MGI) according to the manufacturer's instructions. The DNA sequence was analyzed based on the DNB prepared above using DNBSEQ-G400 (MGI) under conditions of 2 x 200 bp. The results are shown in Table 5-1 and Table 5-2.

Analysis was performed using the DNB sequence information according to the following procedure. First, FastQC1 v0.11.8 was used to check the quality of the raw paired-end read sequence data, and then Cutadapt2 v3.4 was used to trim the DNBSEQ adapter sequence. Further trimmed read sequences were assembled using SPAdes3 v3.12.0. The above quality checking, trimming, and assembling operations were performed on a local server of Galaxy 4 (Samsung). Gene prediction and functional annotation work was performed using Funannotate5 pipeline v1.8.10.

When we conducted an amino acid homology search using the BLAST search system using the above SEQ ID NO: 3 for the amino acid sequence of the gene predicted based on the information on the whole genome sequence of the D138 strain derived from the above method, we found that the homologous “FUN_009158-T1 FUN_009158” was presented as 100% gender. The presented amino acid sequence and DNA sequence are shown below, respectively. The underline in the amino acid sequence indicates a sequence with 100% homology to SEQ ID NO:3.

N-terminal side MFSSAHLAFAAAAAAALVAASPVTELEGQTLAKR ETSYGNPDLVTDQ GNRFKLNFGSTDGHPNACPGHYICYISKNGAQHGDVSLGNPDDFVDEGNRWA (SEQ ID NO: 5)

5'-ATGTTTTCCTCTGCTCATCTCGCTTTCTTTGCTGCTGCTGCTGCCGCTCTTGTTGCTGCAAGCCCTGTCACTGAGCTCGAAGGACAGACTCTTGCCAAGCGCGAGACGTCATACGGCAAT CCCGACTTGGTAACTGACCAGGGCAATCGCTTCAAGCTCAACTTTGGCAGCACCGACGGCCACCCGAACGCGTGTCCCGGACACTACATCTGCTACATCTCAAAGAACGGAGCCCAAACATGGCGA CGTCTCTCTCGGCAATCCCGACGACTTTGTAGATGAGGGCAACCGTTGGGCAA-3' (SEQ ID NO: 6)

This result shows that the DNA sequence encoding the novel anti-fungal polypeptide is predicted in the contig sequence within the whole genome information of the D138 strain, and that the D138 strain encodes the gene that produces this anti-fungal polypeptide. It was shown that it definitely has. The present anti-filamentous fungal polypeptide is secreted from Rhizoctonia solani, but is not limited to secretion from Rhizoctonia solani D138 strain.

[Example 3] Analysis of cDNA sequence of 10 kDa anti-filamentous fungal polypeptide (RNA preparation from cultured bacterial cells of strain D138)
For cDNA analysis of the 10 kDa anti-filamentous polypeptide, RNA was prepared from Rhizoctonia solani strain D138 as follows. Rhizoctonia solani strain D138 was cultured according to the culture method using bran as a solid medium described in Patent Document 1, which is the culture condition for preparing the secreted substance sample from which the 10 kDa anti-filamentous fungal polypeptide was obtained. MN Bead Tubes Type C and NucleoSpin (registered trademark) RNA Plant and Fungi kit (both manufactured by MACHEREY-NAGEL) were used from the bran culture in which mycelium grew on the 2nd, 6th, and 9th day of main culture. An RNA solution was prepared according to the manufacturer's instructions (Table 6).

(Analysis of 3'-side sequence of 10 kDa anti-filamentous fungal polypeptide cDNA by 3'-RACE method)
The Rapid Amplification of cDNA Ends (RACE) method was used to analyze the cDNA sequence, and the cDNA sequence was analyzed in two steps: analysis of the 3'-side sequence using the 3'-RACE method, and analysis of the 5'-side sequence using the 5'-RACE method. The full-length cDNA sequence was analyzed.

First, the 3'-side sequence was analyzed using SMARTer (registered trademark) RACE5'/3' Kit (manufactured by Takara Bio USA) according to the manufacturer's instructions. Using 0.7 μg, 0.9 μg, and 1 μg of total RNA obtained on the 2nd, 6th, and 9th day of culture obtained in the above “RNA preparation from cultured cells of strain D138,” 3'-RACE-Ready 1st-strand cDNA was synthesized according to the instructions.

Next, 3'-RACE PCR was performed according to the instructions using each of the synthesized 3'-RACE-Ready cDNAs as a template. The primer set used the Universal Primer Mix included in the kit and the primer P10-N1-F (SEQ ID NO: 7), which was synthesized based on the N-terminal amino acid sequence information of the 10 kDa anti-fungal polypeptide obtained in Example 2 above. The reaction conditions were [94°C, 1 minute → (94°C, 30 seconds → 72°C, 2 minutes) for 5 cycles → (94°C, 30 seconds → 70°C, 30 seconds → 72°C, 2 minutes) for 5 cycles. Cycles → (94°C, 30 seconds → 68°C, 30 seconds → 72°C, 2 minutes) were carried out for 25 cycles].

Primer P10-N1-F: 5'-GATTACGCCAAGCTTGAGACGTCATACGGCAATCCCGA-3' (SEQ ID NO: 7)

As a result, in the 3'-RACE PCR reaction using any 1st-strand cDNA derived from the bran culture of the D138 strain on the 2nd, 6th, or 9th day as a template, approximately 450 bp of An amplification product was observed, and base sequence analysis revealed the region encoding the C-terminal side of the 10 kDa anti-filamentous fungus polypeptide and the sequence up to the polyA tail (see Figure 10).

(Analysis of 5'-side unknown region sequence of 10kDa anti-filamentous fungus polypeptide cDNA by 5'-RACE method)
Using the approximately 450 bp sequence on the 3'-side obtained by the above-mentioned "Analysis of the 3'-side sequence of the 10 kDa anti-filamentous fungal polypeptide cDNA by the 3'-RACE method", the unknown region sequence on the 5'-side was The analysis was performed using 5'-Full RACE Core Set (manufactured by Takara Bio Inc.) according to the manufacturer's instructions.

0.9 μg, 1.1 μg, and 1.2 μg of total RNA obtained on the 2nd, 6th, and 9th day of culture obtained in the above “Preparation of RNA from cultured bacterial cells of strain D138” were used. 1st-strand cDNA using a 5' end phosphorylated RT-primer (P10-3end_3-RP: SEQ ID NO: 8) prepared based on the known sequence of the 10 kDa anti-filamentous fungal polypeptide cDNA obtained by the 3'-RACE method. was synthesized.

5' end phosphorylated primer P10-3end_3-RP: 5'-(P)GGGAAATGACGAGTT-3' (SEQ ID NO: 8)

Next, after the hybrid RNA was degraded by RNaseH treatment, the single-stranded cDNA was circularized (or concatemerized) by T4 RNA ligase. Using this as a template and using primers prepared in the known region, the unknown region on the 5' side was amplified by inverse PCR. TaKaRa Ex Taq was used as the PCR enzyme, and the 1st PCR was performed using the primer set P10-D59-F (SEQ ID NO: 9) and P10-G47-R (SEQ ID NO: 10) under the reaction conditions [94°C, 3 minutes → (94°C, 30 seconds → 68°C, 30 seconds → 72°C, 1 minute) for 25 cycles → 72°C, 3 minutes].

Primer P10-D59-F
5'-ATGAGGGCAACCGTTGGCGCTG-3' (SEQ ID NO: 9)

Primer P10-G47-R
5'-CCATGTTGGGCTCCGTTCTTTGA-3' (SEQ ID NO: 10)

Furthermore, 1 μL of the stock solution or 10-fold dilution of the 1st PCR reaction solution was added as a template to a 25 μL reaction system, and the 2nd PCR was performed using the following 5 types of primer sets A to E under the reaction conditions [94°C, 3 minutes → (94°C, 30 seconds → 68°C, 30 seconds → 72°C, 1 minute) for 30 cycles → 72°C, 3 minutes].

5 types of primer set A: P10-D59-F/P10-S23-R (SEQ ID NO: 11)
B: P10-D59-F/P10-F17-R (SEQ ID NO: 12)
C: P10-Y82-F (SEQ ID NO: 13)/P10-G47-R
D: P10-Y82-F/P10-S23-R
E: P10-Y82-F/P10-F17-R

P10-S23-R
5'-GCTGCCAAAGTTGAGCTTGAAGC-3' (SEQ ID NO: 11)
P10-F17-R
5'-AAGCGATTGCCCTGGTCAGTTAC-3' (SEQ ID NO: 12)
P10-Y82-F
5'-CATCTGCTATATCAGCAAGTGAAC-3' (SEQ ID NO: 13)

As a result, in a sample in which a series of reactions were performed using total RNA on day 9 of bran culture, in the 2nd PCR reaction using each of the primer sets A to E above, A: approximately 340 bp, B: approximately 320 bp, Amplification products of C: about 340 bp, D: about 270 bp, and E: about 250 bp were obtained. As a result of base sequence analysis, each PCR amplification product encodes the 5'-side unknown region of the 10kDa anti-filamentous fungus polypeptide cDNA, and together with the above 3'-RACE analysis results, it was found that each PCR amplification product encodes the 5'-side unknown region of the 10kDa anti-filamentous fungus polypeptide cDNA. The full length was clarified (see Figure 10). The underlined parts in this sequence indicate the sequences revealed by the 5'-RACE method, and the other parts show the sequences revealed by the 3'-RACE method.

Full-length sequence of 10kDa anti-fungal polypeptide cDNA (SEQ ID NO: 2)
5'- ATCCAACAGATCTCGTCTCAAGCTCACTTTCCAAGCACACACCAACTCAGCCATGTTTTCCTCTGCTCATCTCGCTTTCTTTGCTGCTGCTGCTGCCGCTCTTGTTGCTGCAAGCCCTG TCACTGAGCTCGAAGGACAGACTCTTGCCAAGCGCGAGACGTCATACGGCAATCCCGACTTG GTGTCCCGGACACTACATCTGCTACATCTCAAAGAACGGAGCCCAAACATGGCGACGTCTCTCTCGGCAATCCCGACGACTTTGTAGATGAGGGCAACCGTTGGCGCCTGAACTATGGTAGCACCG ATGGACACCCGAACGCCTGGACACTACATCTGCTATATCAGCAAGTGAACTTATCAACTTAACTCGTCATTTCCCGCAGTTTCCTTTATGTTTGTATTACTTCTCCAGTTAAATCAACCAT AGGAAATATAGCTATCTTTAACACCAAAAAAAAAAAAAAAAAAAAAAAA-3'

As a result of performing a BLAST search on the full-length sequence of the 10 kDa anti-filamentous fungal polypeptide cDNA, excluding the 3' polyA portion, Rhizoctonia solani uncharacterized protein (RhiXN_09327), partial mRNA (Access on number: XM_043329143.1) and 93 .95%, Rhizoctonia solani uncharacterized protein (RhiXN_09328), and partial mRNA (RhiXN_043329144.1), 99.12%, respectively.

Furthermore, the full length amino acid sequence, 122 amino acid residues, of the 10 kDa anti-filamentous fungus polypeptide revealed from the above cDNA sequence is shown in SEQ ID NO: 14. The mature protein after the N-terminal amino acid sequence (SEQ ID NO: 3 above) of the purified protein, excluding the sequence presumed to be the signal peptide (underlined part in SEQ ID NO: 14), consists of 88 amino acid residues, and the estimated molecular weight is It was 9648.24.

Amino acid sequence of 10kDa anti-filamentous polypeptide Full-length N-terminal side MFSSAHLAFFAAAAAAALVAASPVTELEGQTLAKR ETSYGNPDLVTDQGNRFKLNFGSTDGHPNACPGHYICYISKNGAQHGDVSLGNPDDFVDEGNRWRLNYG STDGHPNACPGHYICYISK (SEQ ID NO: 14)

[Example 4] Construction of expression system for recombinant 10kDa anti-filamentous fungus polypeptide and activity of inhibiting growth of filamentous fungi by the recombinant protein (Construction of plasmid for expressing 10kDa anti-filamentous fungus polypeptide)
A plasmid for expressing a 10 kDa anti-fungal polypeptide was constructed based on pET-15b (Novagen; T7 promoter control, ampicillin resistance), a commercially available protein expression vector.

First, a DNA fragment containing a 10 kDa anti-filamentous fungus polypeptide gene with an initiator methionine added to the N-terminus of a mature protein of 88 amino acid residues and its downstream region was amplified by PCR under the following conditions. Using the 3'-RACE-Ready cDNA synthesized in the above [Example 3] "Analysis of the 3'-side sequence of the 10 kDa anti-filamentous fungal polypeptide cDNA by the 3'-RACE method" as a template, the primer iFpET_p10(Met)- Using Fw (SEQ ID NO: 15) and iFp10(end-2)_pET-Rv (SEQ ID NO: 16), the reaction conditions [94°C, 1 minute → (94°C, 30 seconds → 72°C, 2 minutes)] were changed to 5. Cycle → 5 cycles of (94°C, 30 seconds → 70°C, 30 seconds → 72°C, 2 minutes) → 25 cycles of (94°C, 30 seconds → 68°C, 30 seconds → 72°C, 2 minutes)] was carried out. The obtained approximately 300 bp DNA fragment was purified using NucleoSpin Gel & PCR Clean-up Kit (manufactured by MACHEREY-NAGEL), and then inserted into pET-15b vector cut with restriction enzymes NcoI and NdeI using In-Fusion HD. Cloning was performed using a Cloning Kit (manufactured by Clontech). As a result, a plasmid pEp10 for expressing a 10 kDa anti-filamentous fungus polypeptide in which an initiating methionine residue was added to 88 residues of the mature protein was obtained. FIG. 11 and SEQ ID NO: 17 show the insert and surrounding sequences of the 10 kDa anti-fungal polypeptide expression vector.

Primer iFpET_p10(Met)-Fw:
5'-(AGGAGATATA CCATG)G AGACGTCATACGGCAATCCCGA-3' (SEQ ID NO: 15) (underlined: restriction enzyme NcoI recognition sequence, ATG in this sequence is the start methionine codon)

Primer iFp10(end-2)_pET-Rv:
5'-(GGATCCTCGAG CATA)TG GAAAACTGCGGGAAATGACGAGTT-3' (SEQ ID NO: 16) (underlined: restriction enzyme NdeI recognition sequence)

In SEQ ID NO: 15 and SEQ ID NO: 16, the sequence in parentheses indicates a 15 base sequence required for In-Fusion cloning.

(Expression of recombinant protein in E. coli)
E. coli Rosetta (DE3) strain transformed with plasmid pEp10 or pET-15b prepared above was transformed into 20 mL of 2×YT medium (1.6% tryptone, 1.0% tryptone, 1.0% yeast extract, 0.5% NaCl) at a temperature of 30°C and 120 rpm. Turbidity at wavelength 660 nm O. D. When 660 became 1 to 1.1, a portion (200 μL) of the culture solution was collected and the bacterial cells were collected (expression induction time: 0 minutes). Add isopropyl-β-thiogalactopyranoside (IPTG) to the remaining culture solution to a final concentration of 0.2 mM, continue culturing for 90 minutes to induce expression of the recombinant protein, and then add 200 μL of the culture solution. At the same time, all the bacterial cells in the remaining culture solution were collected by centrifugation at 3000×g, 6°C, for 15 minutes, and stored at -80°C.

Bacterial cells from each culture solution collected at 0 and 90 minutes of expression induction time were resuspended in 20 mM sodium acetate buffer (pH 5.2) and then treated in the presence of SDS and mercaptoethanol at 100°C for 5 minutes. The total bacterial protein in the resulting solution was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at a gel concentration of 12.5%, and the results are shown in FIG.

In the pEp10 transformed strain, a prominent protein band was detected at a position of approximately 10 kDa, which was not seen before induction (0 minutes) or in the pET-15b transformed strain, at an expression induction time of 90 minutes, and a 10 kDa antibody was detected. It was confirmed that the filamentous fungal polypeptide recombinant protein was expressed (Figure 12).

(Filamentous fungus growth inhibition activity by genetically recombinant 10 kDa anti-filamentous fungus polypeptide)
In the above-mentioned "Expression of recombinant protein in E. coli", bacterial cells collected 90 minutes after expression induction with IPTG and stored at -80°C were cultured using O. D. The suspension was resuspended in 20 mM sodium acetate buffer (pH 5.2) so that 660 = 20, and ultrasonically disrupted using an ultrasonic generator UD-211 (manufactured by Tomy Kogyo Co., Ltd.). The fungal cell disruption solution was centrifuged at 3000×g, 6° C. for 10 minutes, and the resulting supernatant was sterilized by filtration with a 0.2 μm filter. Using this solution, the filamentous fungus growth inhibitory activity was measured.

The filamentous fungus growth inhibitory activity was measured by the agar-well method using a PDA assay plate as described in [Example 1] above. The results of using Fusarium fujikuroi MAFF235949 strain as the test filamentous fungus are shown in FIG. 13(A).

In the PDA assay plate shown in FIG. 13(A), strong mycelial growth inhibitory activity was confirmed in the supernatant of the ultrasonic disruption solution of the pEp10 transformed strain (FIG. 13(A), pEp10). In addition, when observing the tip of the growing hyphae closest to each well using a phase contrast microscope (Fig. 13(B)), it was found that buffer solution or the supernatant of the ultrasonic disruption solution of the pET-15b transformed strain was added. While hyphae were elongating well near the wells (Figure 13(B), Buffer and pET-15b), hyphal elongation was significantly inhibited near the wells to which the pEp10 transformant strain supernatant was added. (Fig. 13(B), pEp10).

From these results, it was confirmed that the recombinant 10 kDa anti-filamentous fungus polypeptide expressed in Escherichia coli had hyphal growth inhibitory activity of filamentous fungi.

The polypeptides and polynucleotides of the present invention can be used as novel substances with anti-fungal activity, and can be used as compositions for controlling plant-borne fungi such as agricultural chemicals, and as therapeutic agents for infectious diseases in humans and animals. It may be possible to use it as

Claims (7)

A polypeptide having an amino acid sequence represented by SEQ ID NO: 1, or a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and having anti-filamentous activity. A polypeptide having an amino acid sequence represented by SEQ ID NO: 1, or a polypeptide having an amino acid sequence having 70% or more sequence identity with the amino acid sequence, and having anti-filamentous activity. A polynucleotide having the 155th to 418th base sequence of the base sequence represented by SEQ ID NO: 2 or a polypeptide having a base sequence with 70% or more sequence identity with the polynucleotide and having anti-filamentous activity. polynucleotide. An expression vector comprising the polynucleotide according to claim 3. A derivative or modified form of the polypeptide according to claim 1 or 2. A composition for controlling plant-transmissible filamentous fungi, comprising the polypeptide according to claim 1 or 2, a derivative thereof, or a modified product thereof. A pesticide comprising the polypeptide according to claim 1 or 2, a derivative thereof, or a modified product thereof.