JPH05508423A - Novel anti-pathogen peptides and compositions containing the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Novel anti-pathogen peptides and compositions containing the same The present invention provides novel anti-pathogen peptides and compositions containing the same. peptide, a DNA sequence encoding the peptide, a vector comprising the DNA sequence, DNA encoding this peptide! Transgenic plants containing columns, antipathogenic Compositions containing plant peptides and said peptides for controlling plant pathogens METHODS OF USE OF THE COMPOSITION. Method for producing an anti-pathogen peptide or a composition containing the same methods and production of transgenic plants capable of synthesizing anti-pathogen effective amounts of the peptides. Methods of delivery are also included.

Peptides exhibiting antipathogen and especially antimicrobial activity are already known in the art. . Of particular importance are so-called lytic peptides, some of which are found in a wide range of organisms. others have little or no effect. It is known to be fruitless. Many lysates of animal, plant, insect and microbial origin Peptides, such as mammalian defensins ssecurobin ( cecropfns), thionins, melittin (melli tfs), insect differential engine, magainin (a+againfns), me (attacjr+s), dibuterins (dipterins) % Sapesi:/ (sapecfns), caeruHns, xenopsin ( xenopsins) or their hybrids. Has antimicrobial activity The amino acid sequences of several lytic peptides are disclosed in WO39104871. . Lehrer et al. (1986) reported that the human neutrophil differential engine is structurally homologous to the human neutrophil differential engine. Eight antimicrobial peptides (AMPs) from rabbit granule cells have been disclosed.

Eight of the rabbit AMPs (NP-1, NP-2 and NP-8a) are associated with Candida It has been shown to be effective against C. albicans ((:andida albicans). It is shown.

Another class of peptides with anti-pathogen activity are hydrolytic enzymes such as chitinases and and β-1,8-glucanase are typical examples, and they inhibit the growth of fungi. is known (Schlumbaum et al., 1986; Mauch et al., 1 988).

Another class of peptides that have been found to be active is provided. This type of peptide Including known and new representative species. These peptides are used to control plant pathogens. It is particularly suitable for use in anti-pathogen compositions.

Therefore, one of the objects of the present invention is to for controlling plant pathogens containing an anti-pathogen effective amount of the peptide according to the present invention. An object of the present invention is to provide an anti-pathogen composition.

Another object of the present invention is to provide an anti-pathogen-active amount of the peptide or peptide according to the invention. applying to the plant or plant pathogen habitat an anti-pathogen composition comprising: attack of plant pathogens, preferably pathogenic bacteria and/or fungi; and particularly preferably to provide a method of protecting plants from attack by pathogenic bacteria. That's true.

Another object of the invention is that The purpose of the present invention is to provide novel anti-pathogen peptides.

Another object of the present invention is to provide an anti-pathogen peptide according to the present invention and a composition comprising the peptide. An object of the present invention is to provide a method for producing a product.

Yet another object of the invention is to provide one or more peptides of the invention to plant cells. Provides transgenic plants with anti-pathogen peptides based on expression in It is to be.

Another object of the invention is to obtain a DNA sequence encoding a peptide of the invention. The objective is to provide transgenic plants.

Peptides that can be used according to the invention have the following amino acid sequences: (a) Sequence number = 1 or ('b) Sequence number: 2 or (C) Sequence number 20 or (6) N-terminal amino acid sequence substantially homologous to at least one of the above sequences characterized by containing a hydrophobic region at its N-terminus, preferably consisting of one of Ru.

The peptides of the invention can be found in many sources including microorganisms, fungi, plants and higher animals. but are not limited to. They are cell walls, cells It can be obtained from both membrane and cytosol sources.

It has an amino acid sequence in N, 4i:m that is substantially homologous to the above N-terminal sequence; Peptides that can be used according to the invention therefore include, for example, phospholipid transfer proteins. Quality includes, for example, those of the following origins: maize (Tchang et al.

1988; 5ossountzov et al., 1991), spinach (Bo uill.

n et al., 1987; Bernhard et al., 1991), Torres et al. - Schumann et al., 1992), plants and microorganisms (Yamada et al., 1992), carrot (Sterk et al., 1991), finger millet (Cam pos and Rfchardson et al., 1984), and Ricinus (Ri ctnus) (Takishfma et al., 1986). Other examples are putative barley and It may be a small polypeptide isolated from millet millet cells (Mundy and Rogers, l986; 5vensson et al.

1986; Bernhard et al., 1989).

However, preferably the peptides obtained from the leaves of depleted barley plants are Preferred within the scope of the specification and the following properties: (a) Apparent molecular weight Q in the range of approximately 5 kD to 12 kD) retention times of 96.8 and 106.30 minutes, respectively. (Amino l! composition shown in Table 1 below C1 (ω preferably the following amino acid sequence) :(dυ Sequence number: 1 or (d,) Sequence number = 2 or (d,) Sequence number: 6 a hydrophobic N-terminal sequence consisting of one of (e) Especially Corynebacterium cepedonicum (CorynebacteBu It is characterized by its antimicrobial activity against M. sgptdonicum).

The peptides of the invention may also be obtained from other sources, such as the cytosolic fraction; Needless to say, it is not only obtained from the leaves of depleted plants.

a functional derivative of one of the above peptides or a functional hybrid thereof; Good synthetic peptides are also useful as peptides of the invention.

Peptide having the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 4 or SEQ ID NO: 5 Putido is most preferred.

The above peptides are useful for practical purposes, especially in phytopathogenic bacteria and fungi. Surprisingly, it was found to have a very useful microbicidal spectrum against Ta.

For example, the peptides mentioned above have very useful curative, prophylactic and systemic phytotherapeutic properties. can be used to grow and protect cultivated plants. Using these peptides, Inhibits or eradicates plant pathogens that occur in or on plants or on parts of plants. It is possible to destroy it.

Therefore, the present invention also provides the following amino acid sequence 2(a) SEQ ID NO: 1 or ) Sequence number: 2 or (C) Sequence number = 6 or (6) N-terminal amino acid sequence substantially homologous to at least one of the above sequences The above peptide 1 comprising a hydrophobic region at its N-terminus, preferably consisting of one of Seeds or more as active ingredients in anti-pathogen effective amounts are commonly used in agricultural peptide preparations. anti-pathogen compositions, together with suitable carriers and/or auxiliaries for use.

Peptide having the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 4 or SEQ ID NO: 5 Most preferred are compositions containing putide as an active ingredient.

In addition to the active ingredient, the anti-pathogen composition of the present invention contains an agriculturally acceptable carrier and and/or auxiliaries, such as those known in the field of agricultural compositions and where appropriate. In the case of facepieces or liquid auxiliaries, solvents, surfactants and their usual use in agricultural formulations, Items that may include one or more compounds selected from the group consisting of other compounds quality or combination thereof.

Another object of the present invention is to provide anti-pathogenic agents together with suitable carriers customary for agricultural peptide formulations. containing an effective amount of an active ingredient, the active ingredient being a peptide and a lytic peptide according to the present invention. An object of the present invention is to provide an anti-pathogen composition comprising: Each of the active ingredients has a synergistic effect. Compositions that are present in effective amounts are preferred.

Another object of the present invention is to provide an anti-disease agent with appropriate carriers customary for agricultural peptide formulations. Contains a bulk effective amount of an active ingredient, wherein the active ingredient is a phosphoribotransfer protein and a soluble An object of the present invention is to provide an anti-pathogen composition comprising bacterial peptides. Each of the active ingredients Compositions in which they are present in synergistically effective amounts are preferred.

A preferred lytic peptide is thionin.

"Anti-pathogen effective amount" of a substance or composition as used herein When applied to plants or expressed in transgenic plants , meaning the amount that will kill or inhibit the growth of the target plant pathogen. do.

The term "plant pathogen" includes Class 11, bacteria, nematodes and insects.

Preferred target pathogens according to the invention are fungi and bacteria. Bacteria Particularly preferred as target pathogens.

As used herein, substantial sequence homology refers to nucleotide or Refers to the close structural relationship between sequences of amino acids. For example, substantially homologous NA The sequence is 60%, preferably 80% and most preferably 90 or 95% or more, and substantially homologous amino acid sequences are preferably 8 They may be 5% homologous, more preferably 50%, most preferably 50% or more homologous. same may also include deletions of one or more secondary sequences of nucleotides or amino acids. secondary sequences with additional nucleotides or amino acids are present. Includes relationships that are

As used herein, the term "homologous" refers not only to structural homology ( , also encompasses functional homology.

As used herein, the term "structural J" refers to Regarding existing peptides.

The term "inducible" refers to plants that have been exposed to some oppressive environment or external stimulus. A peptide that exists only in plant cells, and that also produces peptides. or peptides whose presence is activated or increased thereby.

Induction can be achieved by chemical means, e.g. acting as an inducer of pathogen-associated proteins in plants. Ethylene, benzoic acid, salicylic acid, and polypropylene Acrylic acid and substituted derivatives thereof may also be used. Induction can also be used to physical and physical means, such as high or low temperatures, physical injury or other Any known inducing means may be used.

The peptides according to the invention can be applied to suitable plant parts, such as depleted plants, in particular depleted barley plants. Can be prepared from a portion of an object.

at a low temperature, preferably 4°C, to minimize protein damage or loss. work and avoid excessive exposure to oxygen. Furthermore, a Various chemicals can be added to various buffer solutions used in purification operations. these For example, protein stabilizers such as glycerol, sulfhydryl reagents such as Dithiothreitol and metal catalysts to minimize oxidation of stain residues. rate of protein exposure to heavy metal ions that can inactivate the protein, e.g. Contains EDTA for prevention. Proteins due to degradation by proteases To avoid loss of texture, the addition of protease inhibitors is often beneficial.

The preparation of the peptides according to the invention is well known in the art (using known separation methods, e.g. precipitation). , phase separation, chromatography, electrophoresis, centrifugation, ultrafiltration, etc. I can.

Precipitation methods include, for example, ammonium sulfate precipitation, acetone precipitation, and polyethyleneimine precipitation. [Borimine P) precipitation and isoelectric precipitation may be included.

Phase separation includes, for example, the use of polyethylene glycol. polyethylene green When a suitable mixture of cole (PEG), salt and water is mixed, a two-phase system is created. Jiru.

The distribution of protein between the two phases depends on the salt and polymer concentrations used, and the temperature. and can be influenced by pH.

Among the chromatography methods known in the art, the following plan purification operations: It can be said that it is useful for: ion exchange chromatography, hydrophobic chromatography affinity chromatography, immobilized metal affinity chromatography chromatography (IMAC), immunoaffinity chromatography, chroma focusing chromatography, gel filtration chromatography and especially Reversed phase fractionation, hydrophobic affinity chromatography, gel filtration or ion exchange High-performance liquid chromatography that can perform either conversion chromatography or high pressure - (HPLC). Columns used in purification operations are preferably defatted or is made as small as possible by the presence of a mild non-ionic detergent, e.g. NP40, in the buffer solution. to avoid loss of protein due to non-specific adsorption to the column material.

In a preferred embodiment of the invention, the coleoptiles or shoots of the assimilated barley plant are removed. removed and strained (used as starting material for protein extraction). The plant material is first ground and powdered with liquid nitrogen, then homogenized, and then Extract with appropriate buffer solution. One or more further extraction steps extract the cell walls and Followed by extraction with a high salt solution to extract membrane proteins. solid The particles are spun down, the supernatant retained and dialyzed against distilled water.

Alternatively, even if the supernatant is desalted by column chromatography, ammonium sulfate It may also be precipitated by nitrogen. Final purification is performed by HPLC.

Determination of the relative ratio of amino acids in the amino acid composition of the peptide according to the present invention is a skill in the art. Methods known in the art, such as the post-column O-phthaldialdehyde method (B enzon and Hare, 1975) or Bidling after total hydrolysis. Pre-column phenylisothiocyanate method according to O+Eyer et al. (1984) This can be done by using

N-terminal amino acid sequencing can be performed by standard methods, e.g. automated Edman degradation. .

Based on this sequence information, identification of the corresponding gene encoding the peptide and Oligonucleotide probes that can be used for isolation can easily be made +W.

The genetic code is known to be degenerate, so different codons are often can be used for one and the same amino acid in With few exceptions, A given amino acid sequence is generally the entire set of oligonucleotides that are similar to each other. can be encoded by the system. However, one of the oligonucleotides in that system Care should be taken to ensure that the injury actually matches the corresponding sequence of the gene being searched for. I have to do it. To limit the number of possible oligonucleotides from the beginning In addition, Lath takes into account the frequency with which a particular codon is actually used in eukaryotic cells. , 8 et al. (191115) may apply. . To facilitate the detection of the desired gene, the DNA probe is may be labeled with a releasable group.

The present invention also provides peptides or fragments or fragments of peptides according to the present invention. and a vector containing the DNA sequence. D.N. The A sequence may be a synthetic NA sequence prepared by methods well known in the art. stomach.

The methods of the present invention can be obtained from a suitable source, such as a cDNA or genomic DNA library. A gene encoding one of the peptides is isolated and the induced gene expression to prepare transgenic plants that are protected from attack by plant pathogens. The gene described above can be easily used.

The invention also includes a DNA sequence encoding at least one peptide of the invention. Concerning transgenic plants.

Moreover, the present invention a) a tract containing a recombinant DNA sequence encoding one or more peptides; produce susgenic plants, or b) two species comprising recombinant DNA sequences encoding one or more peptides; or more transgenic plants and using conventional breeding techniques. cross-breeding the plant with A peptide capable of synthesizing an effective amount of one or more peptides consisting of The present invention relates to a method for producing susgenic plants.

The method preferably comprises a first peptide comprising a recombinant DNA sequence encoding the first peptide. 1 homozygous transgenic plants encoding the second peptide. A second homozygous transgenic plant containing the 8th peptide DNA sequence was created and the 8th peptide A third homozygous transgenic plant containing a recombinant DNA sequence encoding and then cross-breeding eight homozygous plants using conventional breeding techniques. It will be.

In transgenic plants, the synthesis of peptides is controlled by an inducible gene and an inducible can be induced by the use of an inducible expression system consisting of sex regulators.

For expression in plant cells, the DNA sequence is co-transformed in transformed plant cells. one or more promoters or regulators that drive the expression of the coded DNA sequence. in a suitable vector for plant transformation, preferably containing the tropic DNA sequence. Preferably, it is cloned. Many plant expression cassettes and vectors are available in the art. It is well known for. Promoterless constructs can also be induced in plants.

Appropriate control sequences include promoters that are functional in plants and non-translated controls such as 51 and 31. It consists of a clause array.

These sequences can be independently derived from any source, such as a virus, plant or bacterium. can be derived from the A gene. These promoters or regulatory sequences are in their natural configuration elements or can be controlled in their form of expression. Such control is when Intermittent or spatial and expressed and regulated promoters and inducible Includes promoters. Proteins are located in the vacuole or outside the cell, as is well known in the art. It may be expressed using the method described in (EP462065). preferred promo cauliflower mosaic virus containing 35S and 193 promoters Including promoters from. Cauliflower mosaic virus (CaMV) It has been characterized and described by Hahn et al. (1982). This description is incorporated herein by reference. Tubarin synthase, octobin synthase Also preferred are the enzyme and mannovin synthase promoters.

Viral promoters and untranslated sequences suitable for use in plants are functional in plants. and include plant viruses such as cauliflower mosaic virus. do.

CaMV is a unique plant virus that contains double-stranded DNA. At least two types of C aMV transcription promoter, that is, the 19S protein that causes transcription of the CaMV gene The promoter, and the 85S promoter, are functional in plants. 19S promo The 35S promoter and the 35S promoter are preferred plant promoters for use in the present invention. virus promoter.

Plant gene promoters and 5' and 31 non-translated promoters suitable for use in the present invention. An example of translation area is also ribulose bisphosphate carboxylase oxygenator small subunit of enzyme, phosphoenolpyruvate carboxylase, actin, Encoding pathogen-associated proteins and chlorophyll a/b binding proteins This includes genes that are associated with These plant gene regions are equivalent regions from CaMV. (Ma Relli et al., 1985).

Suitable promoters and 5t and 3f untranslated regions from bacterial genes are Includes those present in the T-DNA region of Agrobacterium plasmids.

Some examples of suitable Agrobacterium plasmids are Agrobacterium tu Ti plasmid of A. mefaciens and Ri of Agrobacterium rhizogenes Includes plasmids. Agrobacterium promoters useful in the present invention and 5t and 81 untranslated regions in particular octobin, mannovincin and tuva It is present in the gene encoding phosphorus synthase. These arrays are C The above methods for isolating aMV and plant promoters and untranslated sequences ( Bevan et al., 1983).

In addition to the promoter, the chimeric gene of the invention preferably has a leader at the 51 end. may contain other untranslated sequences called sequences. A suitable leader sequence is 353 Ca Containing leader sequences of various lengths isolated from the MV gene (Pierce et al. (1987), preferred leader sequences are about 50 to about 130 nucleotides. It was isolated from the 85S Ca MV gene, which has a length of .

The expressible DNA, in particular the structural gene to be inserted, has an N-terminus that can function in plant cells. Contains a sequence encoding a terminal signal peptide, or includes a 5-terminal end of such a sequence. Advantageously, they are connected at the ends. The signal peptide is exported via the intramembrane system. It is a transport signal found at the N-terminus of the protein being transported. This signal sequence allows the protein to pass through the endoplasmic reticulum fi first. In the endoplasmic reticulum Once the signal peptide fulfills its function, it immediately separates the protein from the precursor protein. Decompositionally divided.

Due to its specific function, this type of signal peptide sequence In all living cells, whether they are bacterial, yeast, fungi, animal or plant, have been highly conserved during evolution.

Furthermore, the DNA molecules collectively contribute to increasing the ability of vacuole entry. Peptide fragments such as Matsuoka and Naka*ura (1 A propeptide fragment discovered in the N-terminal extension of suboramine by It may consist of another part of the array that encodes the code.

Recombinant DNA consisting of a peptide-encoding DNA sequence is well known in the art. can be introduced into plant cells by many methods, e.g., methods of transforming plant cells. are ultrasonic Joesbo and Brunatedt, 1990), microinfrared The Excidine (Crossway Il, 1986; Neuhaus, 1 987), electrovolesicon (Riggs et al., 1986), intact cells electrovoresicon (Molina et al., 1992), Agrobacterium um-mediated transformation (Hinehee et al., 1988), silicon carbide fiber-mediated transfer (Kaeppler et al., 1990), direct gene transfer (Paszkows Ki et al., 1984), and e.g. Using equipment available from TASS Corporation and DuPont of Ilmington, PLA. (US 4945050; McCabe et al., 1 988; Weissinger et al., 1988; 5anford et al.'1 987 (onion); Christou et al., 1988 (soybean): M cCat+e et al., 1988 (soybean); Datta Ili, 1990 (Rice); Klefn et al., 1988 () Maize); Klein et al., 1 988 () maize); Klefn et al., 1989 (maize); Fromn et al., 1990; Gordon-Kamm et al.

1990 (corn)].

One possible way to introduce genetic material into plant cells is to introduce e.g. contact with a virus or Agrobacterium containing the same DNA.

This can be done by infecting susceptible plant cells or by prototyping derived from plant cells. This can be achieved by co-cultivating the plasts. 111 [within Caliphate] of the present invention The lower mosaic virus (CaMV) is a peptide-encoding DNA according to the present invention. It can be used as a vector to insert the sequence into plants.

Another method for inserting peptide-encoding DNA sequences into cells is to Agrobacterium tumefaciens or Agrobacterium tumefaciens previously transformed with the genetic construct. and/or the use of infection of plant cells with Agrobacterium rhizogenes. . The transgenic plant cells are then cultured under suitable culture conditions known to those skilled in the art. nourished and mossed to form shoots and roots, and the whole plant is eventually formed .

Another possible method of transforming plant material is Pet Agrobacterium rhizogenes or Agrobacterium rhizogenes as described by It et al. (1986) and transformed Agrobacterium tumefaciens. Consists of joint infection.

The peptide-encoding DNA sequences according to the invention can therefore be used, for example, in Agrobacterium Ti plasmid of M. tumefaciens or Agrobacterium rhizogene The Ri plasmid can be transferred into appropriate plant cells.

Ti plasmid or Ri plasmid is transmitted through infection by Agrobacterium It is transferred to a plant and integrated into the plant genome in an appropriate manner.

Both Ti and Ri plasmids are essential for the production of transformed cells. Develop two areas:

One of these regions, the transfer DNA (T-DNA) region, is transferred into plants. and lead to tumor induction.

Other areas of toxicity (vir) are necessary not only for abscess formation but also for its maintenance. It is The size of the transfer DNA region is such that transferability is not impaired. It can be expanded by the addition of peptide-encoding DNA sequences. Eliminate tumor-inducing genes Modified Ti or Ri plasmids are created by removing and adding a selection marker. The vector is a vector for the transfer of the genetic construct according to the present invention into suitable plant cells. It can be used as

The virq region is derived from Agrobacterium cells in which the T-DNA region and vir region are the same. whether present in the same vector within the plant or in different vectors (, Transfer of the Agrobacterium T-DNA region to the bacterial genome occurs. on the chromosome The vir region of also directs the transfer of T-DNA from the vector into plant cells. .

Therefore, in order to transfer peptide-encoding DNA into plant cells, the vfr region and A system in which the and T-DNA regions are located on different vectors can be used. That's it The eel system is known as a "binary vector system" and thereby contains T-DNA. The vector is designated F binary vector J.

Any drug that can be transferred into plant cells and allows selection of transformed cells. T-DNA containing growth vectors such as left border sequence (LB) and right border sequence (RB) from the peptide-encoding DNA sequence according to the invention cloned between and both E. coli and Agrobacterium tumefaciens. Shuttle vectors that can be properly replicated are suitable for use within the scope of the present invention. Ru.

One of the preferred methods of introducing DNA into plant cells by Agrobacterium is , so-called leaf disc transformation using Agrobacterium (Hars h et al., 1985).

Sterilized leaf discs from suitable target plants are coated with peptide-encoding DNA sequences according to the present invention. One of the columns is incubated with Agrobacterium cells and then placed in a suitable nutrient medium. or moved up. solidified by addition of agar and conventional plant growth regulators 1 LS media enriched with species or more are particularly suitable and therefore within the scope of the present invention. preferred within.

a temperature of 20°C to 40°C, preferably 23°C to 35°C, especially 25°C; After several days of incubation, preferably 2 to 3 days, in light and scattered light, the leaf discs become shoots. Transferred to appropriate medium for induction. Does not contain auxin, but instead contains contains tokine, and the selective substance is added depending on the marker gene used. Particularly preferred is LS medium for selecting transformants. Cultures are kept in the light and The cells are transferred to fresh medium at appropriate intervals, preferably at weekly intervals. growing green shoots The shoots are cut and further cultured in a medium that induces the shoots to form roots. auxiliary or cytokines, but the selection agent is used for selection of transformants. Particularly preferred within the scope of the present invention is LS medium supplemented with LS medium.

In addition to Agrobacterium-mediated transformation, plant materials for genetic constructs according to the invention It is also possible within the scope of the invention to use direct transformation methods for insertion into .

Possible methods for direct transfer of genetic material into plant cells include, for example, altering the plasma membrane. Treatment of protoplasts using nucleating procedures, e.g. polyethylene glycol treatment , heat shock treatment, ultrasound or electroporation or any of these operations. (Shillfto et al., 1985).

In electroporation techniques, a polymer consisting of a peptide-encoding DNA sequence is Plant protoplasts or cells along with lasmids are exposed to electrical pulses of high electric fields. be done. This reversibly increases the permeability of biological membranes, resulting in plasmid Insertion becomes possible. Electroborated protoplasts are It creates a new cell wall, divides, and then forms callus tissue. transformed plants Selection of cells can be performed by the phenotypic markers described above.

Based on purely chemical methods and which can be carried out rapidly with very high efficiency, Another method for direct introduction of genetic material into cells is Negrutiu et al. (198 7).

Another means of directly inserting the genetic material contained in the vector into plant cells is e.g. Microinjection (Neu haus et al.

1987) or microprojectile cells coated with transforming DNA. Input into [“Micro Projectile Input J” (fang et al., 19 88)) or by using purely physical methods according to Accelerated by the impact pressure through the DNA-containing solute in the direction of the cell to be exchanged , whereby the solution is finely atomized into a mist as a result of the impact pressure (EP 4346 16).

Screening of plant cells, tissues and plants for the presence of specific DNA sequences The analysis is performed by Southern analysis (Southern, 1975). This person Details of the method are given by Maniatis et al. (1982). This screenplay Coating can also be performed using polymerase chain reaction (PCR). P The CR method is detailed in Mulli S et al. (1987) and Erlich (1989). It is described in.

Transformation of plant cells involves separating transformed cells from non-transformed cells. It includes letting go. One common method of such separation or selection is transformation. containing the selectable marker gene in the material to be inserted into the cells It is. As a result, only successfully transformed cells contain the marker gene Will.

The translation products of the marker genes are then responsible for conferring the phenotypic characteristics that enable selection. Dew. Phenotypic characteristics may be influenced by the presence of certain chemicals, e.g. antibiotics, e.g. in selective media. Survives in the presence of kanamycin, 0418, paramomycin and others added to It is the ability to

Some examples of genes that confer antibiotic resistance are e.g. Transferase [Kanamasin resistance, Velten et al. (1984)); High Gromycin phosphotransferase (hygromycin resistance + van d Kanamaishi derived from Tn5) (NPTII) gene (Bevan et al. (1983); McBride et al. (1990)), the FAT gene described in Thompson et al. (1987) and chloramphenicol acetyltransferase.

Selectable markers in tissue culture for identification of plant cells containing genetically engineered vectors Examples of genes that are primarily useful as enzymes are those that encode enzymes that produce chromogenic substances. It's a legacy. One example is the gene encoding the production of β-glucuronidase (GUS) It is. This enzyme is widely used, and its preparation and use was described by Jeffers on (1987).

Once transformed, plant cells are cultured on selective media, and surviving cells are further studied. selected for investigation and manipulation. Selection methods and materials are well known to those skilled in the art. and the selected cells will be successfully transformed with foreign DNA to a high degree. This makes it possible to select surviving cells by predicting the

For example, transformation of plant cells or plants using Agrobacterium Ti plasmids. Plant cells transformed with the Ti plasmid such that after transformation, the enzyme is expressed. Alternatively, plants can be selected with appropriate phenotypic markers. These phenotypic markers - includes, but is not limited to, antibiotic resistance. Other phenotypic markers are known in the art and can be used in the present invention.

Positive clones are reproduced according to procedures well known in the art. Continuing, shape The transformed plant is characterized by the presence of and/or the expression of desired characteristics. The degree is evaluated. The first evaluation is e.g. bacterial/fungal resistance of the transformed plants. the degree of genetic development, stable inheritance of desired characteristics, field testing, etc.

Plant pathogens that may be targets of the present invention include the following classes: Bacteria (e.g. Corynebacterium, Pseudomonas, Xanthomonas and Eru) fungi, e.g. Deuteromycetes (e.g. Botrytis, Fusarium, Sebtorium): Fungi, e.g. a): Ascomycetes 1I4 (e.g. Erysiphe, Monilia); Oomycetes (e.g. Peronosvora, Phytophthora, Brasmovara, Colletotrichum and Fichiu Basidomycetes (e.g. Rhizoctonia and Bushinia): and insects and nematodes (e.g. Meloidogene, Caenorhabditis, Globola, Heterodes) La and Brachylenchus species). For example, pathogens such as Botrytis cinerea and Coleto Triticum lagenarium, Erysiphe graminis (wheat pathogen), Monilia Fructicola, Peronospora tabacina, Phytophthora parasitica, Brass Mopala biticola, Phytium ultimum (soil pathogen), Rhizoctonia so Contains species of Rani (another soil pathogen) and Cebutria nodorum (a wheat pathogen) It is possible.

Target crops to be protected within the scope of the present invention include the following plant species: corn body, husk w4 (e.g. wheat, barley, rye, oats, rice, sorghum and continuous crops), beets (e.g. sugar beets and fodderbeet), stone fruits, pears Fruits and soft fruits (e.g. apples, pears, plums, peaches, almonds, cheesies, strawberries, raspberries and blackberries), legumes (e.g. beans, cilantro) legumes, peas, soybeans), oil plants (e.g. rape, mustard, hobby, olives, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts), cucumbers Limaceae plants (e.g. cucumber, mallow, melon), fiber plants (e.g. cotton, flax, jute), citrus fruits (e.g. oranges, lemons, grapefruit, mandarin), vegetables (e.g. spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, paprika), Lauraceae (e.g. bocado, cinnamon, camphor), or plants such as tobacco, nuts, coffee, sato Corn, tea, grapes, hops, banana and natural rubber plants, as well as ornamental plants. thing.

The present invention further provides that the peptide according to the present invention is used in a suitable carrier commonly used in agricultural peptide preparations. Antipathogenic agent consisting of homogeneous mixing of body and/or one or more auxiliaries Including the manufacture of body compositions. The active ingredient is one or more of the above compounds or homogeneously mixed with the above group of compounds.

The present invention also provides an active ingredient or an anti-pathogen composition containing an active ingredient in plants or plants. protection of plants from attack by plant pathogens, consisting of application to plant pathogen habitats; It also relates to how to

In one embodiment, the peptides of the invention typically contain one or more agricultural applied prophylactically or therapeutically in the form of a composition with an acceptable carrier and Other compounds may be applied simultaneously or sequentially to the area or to the plants to be treated. . These compounds may be used as fertilizers or micronutrient donors or to affect plant growth. It may be a bosu or other formulation. They are also selective herbicides, insecticides, fungicides, Bactericides, all insecticides, molluscicides or mixtures of these preparations. and, if desired, other carriers, surfactants or application promoters customary in the formulation industry. Auxiliary agents may also be used. Suitable carriers and auxiliaries may be solid or liquid. (and substances customary in the formulation industry, such as natural or recycled minerals, solvents, dispersants, Corresponds to a warming agent, thickener, binder or fertilizer.

Preferable agrochemical compositions containing the active ingredient or at least one active ingredient of the present invention The new application method is eastern application.

The number of applications and the rate of application depend on the intensity of spread by the corresponding pathogen. deer However, the active ingredient can be applied by soaking the liquid composition into the plant area or by applying it to the soil. By applying the compound to the soil in solid form, e.g. in granular form (soil application), It can penetrate the plant through the soil and through the roots (osmotic action). Active ingredients enable seeds Seeds can be prepared by soaking them in a liquid formulation containing the ingredients or by coating the seeds with a solid formulation. may be applied. In special cases, further application forms, e.g. Alternatively, selective treatment of buds is also possible.

The active ingredients can be used in their neat form or preferably together with auxiliaries customary in the pharmaceutical industry. and can be prepared by known methods such as emulsion stock solutions, coatable pastes, direct spraying, etc. or dilutable solutions, wettable powders, soluble powders, dusts, granules, and further e.g. It is formulated into capsules filled with polymeric material. As well as the nature of the composition, the application The method of use, such as spraying, atomization, dispersion, sprinkling, coating, or pouring, depends on the intended target. and application environment. Advantageous rates of application are usually hectares (ha ,) 100g to 5kg of active ingredients (a, f,), preferably 100g ~2kg　a,i,/ha, especially 200g~500g　a,i,/ha It is.

Preparations, compositions or compositions containing the active ingredient and optionally solid or liquid auxiliaries. The formulation can be prepared by adding the active ingredient to a filler, such as a solvent, a solid carrier, etc., by a known method. and, if appropriate, by homogeneous mixing with surface-active compounds (surfactants). be manufactured again.

Suitable solvents or dispersants are primarily diaromatic substances such as toluene. xylenes, benzene and alkylnaphthalenes: chlorinated aromatics and chlorinated Aliphatic hydrocarbons such as chlorobenzene, chloroethylene and methylene chloride; Aliphatic hydrocarbons such as cyclohexane and paraffins, such as petroleum distillates: alco such as butanols and glycols and their ethers and esters. Ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and and cyclohexanone: and strong polar solvents such as dimethylformamide, N-methyl Lu-2- and lolidone, dimethyl sulfoxide or dimethyl formamide, and Epoxidized vegetable oil such as epoxidized coconut oil or soybean oil; water.

Solid carriers used for example in dusts and dispersible powders are typically calcite, talc, calcium Natural mineral fillers like olin, montmorillonite or attapulgite Ru.

Adding highly dispersed silicic acid or highly dispersed absorbent polymers to improve physical properties is also possible. Suitable granulated absorbent carriers are of the porous type, e.g. pumice, crushed brick, seviolite or bentonite; and suitable non-absorbent carriers are It is a calcite or sand-like substance. Furthermore, a large number of granulated minerals and and organic materials, especially dolomite or pulverized plants! A single shell can be used.

Suitable surface-active compounds are nonionic, cationic compounds with good dispersibility and wetting properties. It is an anionic and/or anionic surfactant. The word "surfactant" It will be understood that mixtures of surfactants are also included.

Suitable anionic surfactants are so-called water-soluble soaps and water-soluble synthetic surfactants. The compound can be both.

Suitable soaps are alkali metal salts of higher fatty acids (chains of 10 to 22 carbon atoms); Alkaline earth metal salts or unsubstituted or substituted ammonium salts, such as ole inic acid or stearic acid, or obtained from e.g. coconut oil or tallow It is a sodium or potassium salt of a natural fatty acid mixture. fatty acid methyltauri salts may also be used.

However, so-called synthetic surfactants, especially fatty sulfonates, sulfonated benzimidazole derivatives or alkylaryl sulfones is used more frequently.

Fatty sulfonates or sulfates are usually alkali metal salts, alkaline earth metals in the form of a salt or an unsubstituted or substituted ammonium salt, and in the form of an acyl group C8-C22 alkyl groups which also contain alkyl moieties, e.g. Fats obtained from lignosulfonic acid, dodecyl sulfate or natural fatty acids It is a sodium or calcium salt of a mixture of alcohol sulfates. this These compounds include sulfated and sulfonated fatty alcohol/ethylene oxide additions. Also includes salt.

The sulfonated benzimidazole derivative preferably has two sulfonic acid groups and and one fatty acid group containing 22 carbon atoms. alkylaryl sulfo Examples of nates are dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or naphthalene sulfonic acid/formaldehyde condensation product sodium, calcium salt or triethanolamine salt. the corresponding phosphate, e.g. 4 Phosphoric acid of p-nonylphenol adduct containing from 14 moles of ethylene oxide Salts of esters are also suitable.

The nonionic surfactant is preferably an aliphatic or cycloaliphatic alcohol, or Polyglycol ethers of saturated or unsaturated fatty acids and alkylphenols a derivative having 3 to 30 glycol ether groups, (N aliphatic) 8 to 20 carbon atoms in the hydrocarbon moiety and the alkyl phenol 6 to 18 carbon atoms in the ru moiety.

Other suitable nonionic surfactants are polyethylene oxide and polypropylene. Recall, ethylene diamino polypropylene glycol and 1 in the alkyl chain Water-soluble with alkyl polypropylene glycol containing to 10 carbon atoms The adduct contains 20 to 250 ethylene glycol ether groups. and IO to 100 propylene glycol ether groups. these The compound usually contains 1 to 5 ethylene glycol units per propylene glycol unit. Contains units of 1.

Typical examples of nonionic surfactants are nonylphenol polyethoxyethanol, Castor oil polyglycol ether, polypropylene/polyethylene oxide addition substances, tributylphenoxypolyethoxyethanol, polyethylene glycol and and octylphenoxypolyethoxyethanol. Polyoxyethylene Fatty acid esters of Rubitan, such as polyoxyethylene sorbitan triolate are also suitable nonionic surfactants.

Cationic surfactants preferably contain at least one carbon atom as an N-substituent. An alkyl group having 8 to 22 molecules and lower unsubstituted or halogen as other substituents Quaternary ammonium containing alkyl group, benzyl group or lower hydroxyalkyl group It is a nium salt. The salt is preferably a halide, methyl sulfate or ethyl sulfate. It is in the form of an acid salt, for example stearyltrimethylammonium chloride or and di(2-chloroethyl)ethyl ammonium bromide.

Surfactants customary in the pharmaceutical industry include, for example, McCutcheon's Deter. gents and Bmulsiffers Annual (1979) and 5isely and Wood (1980).

The agrochemical composition contains about 0.1 to about 99% of the active ingredient, preferably about 0.1 to about 99%. about 95%, especially about 3 to about 90%, and about 1 to about 99% solid or liquid auxiliary. 9%, preferably about 1 to about 99%, especially about 5 to about 95%, and surfactant from about 0 to about 25%, preferably from about 0.1 to about 25%, especially about 0.1%. Contains from about 20% to about 20%.

Commercially available products may preferably be formulated as concentrates, but the end user may prefer to prepare diluted products. agents would normally be used.

Another object of the present invention is to include as an active ingredient in the anti-pathogen effective composition according to the present invention. The use of lipid transfer proteins.

Another object of the present invention is to use the present invention as an active ingredient in the anti-pathogen effective composition according to the present invention. Use of the peptide according to the invention. Amino acid sequence below: (a) A, 1a lle Thr Cys Gly Gin Val Ser Ser Ala Leu or (b) Ala　lie　Ser　Cys　Gly　Gln　Val　Ser　S er Ala Lau 5erPro Cys lie Ser Tyr Al a　Arg　Gly　Asn　Asn　Ala(C)Ala　Lie　Ser　 Cys Gly Gin Val Ser Ser Ala Leu 5erP ro Cys lie Ser Tyr Ala Arg Gly Asn G ly Ala (mountain) N-terminal amino acid substantially homologous to at least one of the above sequences acid sequence A peptide comprising a hydrophobic region at its N-terminus, preferably consisting of one of It is preferable to use it as an active ingredient in the anti-pathogen effective composition according to the invention.

Another object of the invention is to apply an anti-pathogen effective amount of the peptide or composition of the invention to plants or plants. the plant pathogen, preferably pathogenic, by applying to the plant pathogen habitat Provided is a method of protecting plants from bacterial and/or fungal attack.

Another object of the invention is to transfer an anti-pathogen effective amount of phospholipids into transgenic plants. The present invention provides a method for controlling plant pathogens, which comprises expressing a transferred protein.

Another object of the invention is to express anti-pathogen effective amounts of the peptides of the invention. The present invention provides a method for controlling plant pathogens.

Another object of the invention consists in expressing an anti-pathogen effective amount of the active ingredient; of plant pathogens whose active ingredients consist of phosphoribotransfer proteins and lytic peptides. This is to provide a pest control method.

Another object of the invention consists in expressing an anti-pathogen effective amount of the active ingredient; The present invention provides a method for controlling plant pathogens, the active ingredient of which is the peptide of the present invention.

Another object of the invention is to: a) Recombinant DNA encoding one or more phospholipid transfer proteins Create a transgenic plant containing the sequence, b) Injecting one or more phospholipid transfer proteins into the transgenic plant. synthesize The present invention provides a method for controlling plant pathogens comprising:

Following stages: a) Peptides shown in SEQ ID NOs: 3 to 5 and substantially homologous thereto A tract containing a recombinant DNA sequence encoding one or more peptides with to create susgenic plants, b)) The peptides shown in SEQ ID NOs: 3 to 5 and synthesize peptides 111 or more that have substantial homology to them; Also included are methods of controlling plant pathogens.

Following stages: a) A trans-transplant containing a recombinant DNA sequence encoding an effective amount of an anti-pathogen active ingredient. Genetic plants are produced, and the active ingredients are peptides shown in SEQ ID NOs: 3 to 5. and peptides and lytic peptides having substantial homology thereto. , b)) Synthesizing an effective amount of an anti-pathogen active ingredient in a transgenic plant; peptides shown in SEQ ID NOs: 23 to 5 and substantially homologous thereto. A method for controlling plant pathogens consisting of peptides containing peptides and lytic peptides. It also includes the law.

The invention also includes the following steps: a) Recombinant DNA encoding one or more phospholipid transfer proteins Create a transgenic plant containing the sequence, b) Injecting one or more phospholipid transfer proteins into the transgenic plant. synthesize The present invention relates to a method for controlling plant pathogens consisting of.

The present invention a) Recombinant encoding phospholipid transfer proteins and optionally lytic peptides create transgenic plants containing the DNA sequence, or b) Recombinant encoding phospholipid transfer proteins and optionally lytic peptides Create two or more transgenic plants containing the same DNA sequence, and and cross-breeding the plant using conventional breeding techniques. The method further includes a method for producing a transgenic plant capable of synthesizing the active ingredient.

The purpose of the present invention is to a) Peptides shown in SEQ ID NOs: 3 to 5 and substantially homologous thereto recombinant DNA sequences encoding peptides and optionally lytic peptides having or b)) in a transgenic manner. Peptides shown in SEQ ID NOs: 3 to 5 and substantially homologous thereto a recombinant DNA sequence encoding a peptide and optionally a lytic peptide. Generate two or more transgenic plants and use conventional breeding techniques. hybridizing the plant using a technique; A transgenic plant capable of synthesizing an effective amount of an anti-pathogen active ingredient consisting of This is to provide a method for producing.

After a general description of the invention, for better understanding, the following are set forth for illustrative purposes only: Specific non-limiting examples are provided below, unless otherwise specified.

Non-limiting operational examples Example 1: Growth of barley plants Barley cultivar Betzes and barley cultivar Bomi in a shallow dish (15x 30 cm x 8 cm) in vermiculate. Fill the dish with sterile water, Then, keep it in 9 places in the cabinet for 7 days. Remove cotyledons and/or shoots Remove, immediately freeze in liquid nitrogen and store at -70°C.

Example 2: Protein purification 2.1: Tissue extraction Approximately 20 g of the fresh weight of the collected offspring and/or shoots was placed in a mortar with liquid nitrogen. Grind into powder using a pestle. Powdered material is 0. IM Tris HC1, 10mM E D.T.A.

Extract with 4 volumes (80 ml) of a buffer solution having a pH of 7.5.

The combined extracts are centrifuged at 5000Xg for 10 minutes at 4°C. Discard the supernatant , and extract the pellets twice (80 ml each time) with distilled water.

2.2 = Cell wall extraction Following extraction with water, a high salt consisting of 1,5M LiCl was formed into pellets in water. Extract with 50 ml of solution. The extraction mixture is kept at 4°C for 1 hour. Next, extract the mixture Centrifuge the material at 5000×g for 10 minutes at 4°C.

retain the supernatant, and (51) Membrane that can maintain a molecular weight of >8000kD (5pectra/P).

r; MWCO: 3000; SPBCTRUM@, Medical Industry Dialysis against distilled water 51 using ) Desalination through a Sephadex G25 [Pharmacia] column, or (Precipitation with C1 solid ammonium sulfate [43.7 g per LOOml supernatant), Do one of the following:

If method (a) or (b) is used, the desalted supernatant is subjected to lyophilization. more concentrated.

2.3 HPLC minutes The concentrated extract (lyophilizate or ammonium sulfate precipitate) as described above is Add 0.1% [w/v] trifluoroacetic acid (TFA) to 1 ml of growing water. lysed and subjected to HPLC fractionation. The chromatography system used is Ultrabore C-4 column (Beckman), 126-distem gold Beckman The detector consisted of a 100-m bomb and a Beckman UV16 low system gold detector.

UV detection is performed at a wavelength of 214 nm. A surface area of 1 m1 or more is poured into the C-4 column. entered. Initially, the mobile phase was 100% distilled water containing 0.1% (w/v) TFA. It is. The flow rate is set at 0.5rnl/min at room temperature. Next, 0 to 80% ( A linear gradient of isopropanol (V/V) takes 180 minutes (=90m l) applied to the column and then 30 to 50% (V/V) of isopropyl A gradient of 100 mm is applied over 15 minutes (=7.5 ml). 3 to tO ml fractions were collected and loaded into a Sabent Speed Vac Concentrate. Concentrate and dry in vacuum in a turret. Retention time: 96.3 minutes [CW18], 104. After 4 minutes 1: cW20), 106.3 minutes (CW21) and 111 minutes (CW22) The eluted fractions are collected and subjected to further analysis. Protein is Pa 1 i n and LowryCLowry et al., 1951) or BAS using protein assay reagents (Pierce, Rockford, IL, USA). It is determined. cwis, CW20. The peaks corresponding to CW21 and CW22 are Collected by hand as a single fraction, the highest peaks are the “rising” and “falling” portions of the peak. Discard the first 20 seconds and the last 20 seconds. The resulting protein fraction has three types of electricity. Uniform by electrophoresis (acidic, basic and 5DS-PAGE) and N*@sequencing It turned out to be.

2.4 Molecular weight The molecular weights of peptides CW18 and CW21 are each 8 as determined by mass spectrometry. ．． 78kD and 8.88kD.

2.5 Amino acid composition In the amino acid composition of peptides CW18, CW21, cwzo and 0w22 The relative proportions of amino acids were determined by Bfdling@eyar et al. after 24 hours of total hydrolysis. (19B4) G: Determined using the compliant pre-column phenylisothiocyanate method. determined.

The results shown in Table 1 show that the amino acids shown for all of the amino acids yielded similar measurements. It should be understood as the respective % (measured value) of acid.

Table 1 = Peptides CW18, CW21, CW2 (l and CW22 (7) amino acid set Synthetic (residues/100) amino acids Cll1/1111 CW21 CW2OCW2 2Cys 6.2 5.6 635.5 Ajx" 3.9 3.9 5.2 5.20Lx" 5.0 5.1 4.6 5.4Ser 11.9 t2. ．． 6 1f, 6 11.4axy 13.3 12.7 13.0 13.3His 1.3 - 1.1 - Arg 6.0 4.1 3.4 3.6Thr 53 1g 3.2 Zj Earth 15.9 19.3 19.3 18.9Pro 6.0 7.9 6.7 6. OTy 3.0 2.1 2.0 2.2 Val 6.4 6.3 6. 4 6.8ne 5.0 F, 1 5.9 5.8LAu 4.9 4.3 4 ．． 6 4.6Lys 5.7 6.2 6.4 7.7Aax is Asn or A sp.

GIX means either Gin or Glu.

2.6 Amino acid sequence Identifies the N-terminal amino acid sequence and the entire amino acid sequence of the peptide of the present invention. Automated Edman degradation was performed using the Applied Biosystems 470A! phase Sequencer (Applied Biosystems, Foss, California, USA) It is carried out using [Tarcity]. PTH amino acid is manufactured by Applied Biosystems Identified using a Muda 120 APTH analyzer.

The amino acid sequence of peptide CW2i is SEQ ID NO: 4. Peptide CW18 The amino acid sequences are SEQ ID NO: 3 (Bomi) and SEQ ID NO: 5 (Bomi), respectively. etzes). N-terminal amino acid sequences of CW20 and CW22 (amino acid 12) is identical to the N-terminal sequence of CW21.

Example 3: Formulation side of tfC pathogenic composition Below, % of composition is % by weight.

Solution: a b cd Vepti l’ SON IO $51 951 Ethylene glycol body mol methyl ether Te# 20% - Polyethylene Glyco-k 400 - 70% - N-Methi Ruby-2-BiOridone - 20% - Epoxidized coconut oil 1% 5% Petroleum content (boiling point range 160-190℃) - 94% - These solutions are in the form of fine droplets. Suitable for application under conditions.

Example 4: Antibacterial assay (a) Microtiter plate shows the inhibitory effect of CW18 and CW21 peptides selected to examine the results. The test substance (5 μg) was dissolved in water and poured into C , sepgdonicuyt strain at a concentration of approximately 10' bacteria/ml. Inoculate in advance and final volume 150μ! 1/3 concentrated N-1 medium (pH 7.2) Ishi 7.4: Difco Beef Extract 1. og; Difco East ・Extract 2.0g; Difco Bactobebutone 5.0g: NaCl3. 0g; and water to make 10100O]. Test substance in medium The final concentration of assumption).

Bacterial growth was determined at 25°C by measuring the absorption of the medium at 492 nm. Evaluation is made after 48 hours of incubation.

For controls, α1/β-thionine from wheat pressure leaves and from barley plan. Two standards are used in the above test: LT26-thioene.

Soluble chemical thioenes are described by Refa+ann-Philfpp et al. (1989). R prepared as described and described above for CWI8 and CW21 Further purification is carried out using a P-HPLC program. Uni-LT2B has 28 residues The sequence deduced from cDNA clone DG3 (Boh lmann and Apel, 1987).

Endocytic thionin is described by Garcia-01medo et al. (1968) obtained by precipitation from a petroleum ether extract as in RP- Purified by HPLC.

The results of the antibacterial assay are shown in Figure 1.

(b) CWI8, CW21, CW2O and CW22 are dissolved in water as required. be understood. C,5epedonicurtr (also known as CLavibactr) michiganesis subspecies 5zpedonicus) and Pseud orxonas soLanacearum each final concentration! O’cfu/ml in a sterile microtiter plate with a final volume of 150 μm (Peptide i00a 1 + Nutrient Bull: 7s 50μ7゜Oxoid). 16 or more at 28℃ After 24 hours of incubation, growth was determined by absorbance at 492 nm in an ELISA plate reader. recorded by measuring the

Table 2: C,stpe of CW18, CW21, CW2O, CW22 and thioene activity against P. donicum and P. soLanacgaruw (EC-5 0 inhibition 4) C, stpedtynicwn P, solaruxtantm? shi/, 17 beak ppm IJM umbrella ppm…”CW21 3 0゜3 5 0.6 CW20 1.5 0.2 CIiV22 (1<0.1 3 0.3+ma=>5 1 5 1 “EC-50: required to inhibit bacterial growth by 50% compared to control Peptide amount 9 Assumed molecular weight 9000 (C) Bacteria are cultured in microtiter plates. Peptide increases Added to the culture medium. Growth as an increase in optical density was measured 2 and 3 days after inoculation. determined.

The starting inoculum consisted of 101 bacteria/ml. The concentration of peptide was 1100pp. be.

Table 8: Inhibition of growth of Xanthowtonas oryzae (control %) CWI8 C11I20 CW21 CW22 Control day 2 73 27 40 39 100 3rd day 77 313 29 39 100 Example 5: Antibacterial type assay (a) Antibacterial assays can be performed similarly to the antibacterial assays described above. N 1 In place of the culture medium, the spores of the fungi to be tested are inoculated in advance with approximately 10 to 100% of spores. Tomato medium is used. The final concentration of test substance in the medium is 20% depending on the test organism. 00 or 200 to 0.2 ppm. Place the plate in a shaker in the dark Evaluations are made after 48 hours of incubation in a wet chamber at 23'C.

Different test solutions are tested 2/3 times on the sample.

Pathogen growth is recorded by measuring medium uptake at 5951 m.

Compared to untreated controls, CWI8 and CW2i are more resistant to various fungal pathogens. Shows antibacterial activity.

(bl　Spores were placed on potato dextrose agar (Difco) plates for 25 minutes. Collected from 8-day-old cultures grown at °C. The spores are tiroekaert et al. (1990) and stored in 20% glycerol at -20°C. be done. In the growth inhibition assay, the spore suspension (10000 spores/ml ) in 67.5 μf potato dextrose broth per microtiter well. Grow at 25 °C and incubate with protein dissolved in water as required ( Total capacity 75 μi). Measurement of the absorption of the culture at 540 nm shows that the fungi within a wide OD range linearly correlated with biomass. After 26 to 44 hours of incubation at 25°C, growth to measure absorption at 540 nm in an Eliza microplate reader. recorded by.

The results are summarized in Tables 4 and 5.

Table 4: Fus of CWI 8, CW21, CW2O, CW22 and thioene Activity against Arium 5oLani (EC-50CW18 45 5 CW21 130 3 CW20 200 20 CW22 100 10 Thionin 10 2 ``VEC-50: required to inhibit bacterial growth by 50% compared to control amount of peptides 0 molecular weight is assumed to be 9000 Table 5: FusariuHsoLan of compositions containing CW21 and thioene Activity against i (EC-50 value) Protein ppm μM・ 10ppm CW2m + Titnin 4 En 0, 7520pHCW21 + Chi Onin 1 0.2” EC-50: Reduces bacterial growth by 50% compared to control Amount of peptide required to inhibit 0 molecular weight is assumed to be 9000 Example 6: Form of Agrobacterium Conversion binary vector is Agrobacterium The transformant is transformed into the S. tumefaciensis strain LB4404 according to the following method. a Globacterium strains were grown in LBMG medium (50% L broth, 50% mannitol-g Lutamate Broth (Garfinkel and Neter, 1980) 5 Grow overnight in ml at 30°C. Add 5ml of culture solution to 250ml of LBM0 and shake vigorously until the culture concentration reaches 0D = 0.6 at a wavelength of 600 nm. move. Cells were collected by centrifugation at 8000Xg and LBMGS Resuspend in ml. 200 μm of cells were incubated with binary plasmid DNA in LBMG. 2 to 1 μg and simultaneously freeze the mixture on dry ice/ethanol. to tie After 5 minutes, place the tube in a water bath at 87℃ for 5 minutes, then add 02ml of LBM. Added. The suspension was kept in a 30°C water bath for 2 to 3 hours, then the cells were centrifuged. Collect by separation. Cells were resuspended in the lowest volume of LBMG and then selective medium ( Blating onto LBMG plate (containing 100 μg/ml gentamicin). Ru. Colonies appear after 2-3 days at 30°C.

Positive colonies were selected and confirmed by Southern blot analysis and plant traits Used for conversion experiments.

Plant tissues are transformed with the vectors described above by any technique known in the art.

Methods used to introduce DNA into plant cells include, for example: to: Agrobacterium tumefaciens in plants, plant tissues or cells direct infection or co-culture of both (Hor3h et al., 1986; Marton, (1984), Paszkowski et al. (1984).

HP 164575; 5hf11ito et al. (1985); Potrykus (1985); Lorz et al. (1985); Fro+am et al. (1987); GB 2140822; and the method described in Negrutiu et al. (1987) Treatment of foreign DNA and protoplasts with polyethylene glycol (PEG) ) with heat retention (Negrutfu et al., 1987), [Refch et al. (1986a and b)], microprojectile stamping (Klein et al., 1987).

A. Tobacco industry disc transformation Adjusted to pH 5,6, 0.15% mannitol, 50°C g/ml kanamycin, Supplemented with 50°C g/ml spectinomycin and 1 mg/ml streptomycin. Grow Agrobacterium for 18 to 24 hours in supplemented glutamate medium. and dilute to a 0D600 of 0.2 in the same medium without antibiotics. Next bacteria Grow for 3 to 5 hours and dilute to an OD600 of 0.2 to 0.4. child were grown under sterile conditions in GA7 vessels according to a modified method of Horsch et al. (1985). Five or more punches punched from the leaves of the xanthi cultivar of Nicotiana tabacum Used for infection of 7 mm discs.

Leaf discs were treated with Murashige and Skoog mass salt and a little salt (MS), Img/ 0.7% containing l benzyladenine and 1 mg/ml α-naphthalene acetic acid Hold on agar for 2 days, 50°C, 7ml kanamycin, 100 u g/m 1 carbenicillin and 100 gg/m! in a similar medium containing meftoxin. Move. The shoots that form on the discs are excised and placed in a GA7 container at 50°C g/m 6 by subculturing the shoot pieces on MS medium containing l kanamycin. Propagate until plantlets are obtained.

50℃g/m without hormones! Rooting the plantlets in a medium containing kanamycin; Transferred to soil, cultivated and acclimated in phytotron for induction treatment with chemical control agents. Transfer to greenhouse. During anthesis, flowers are induced to self-pollinate.

Collect seeds and let them mature.

B.) Production of transgenic tobacco callus and plants Agrobacterium on leaves Used to transform callus formed from discs (A). Kanamai Callus formed on MSBN-containing selective medium was treated with MS high salt, low salt, and MSBN selective medium. Fe-EDTA (Gibco, catalog number 500-1117: 4.3 g/l), M S vitamin, 100mg/l myo-inositol, 20g/l sucrose, 2mg/l The cells were maintained on callus growth medium consisting of naphthalene acetic acid and 0.3 mg/l kinetin. hold

Transfer the callus pieces to MSBN and then perform the method described in A to transduce the Callus can be used to regenerate genic plants.

C. Transformation of carrots Agrobacterium is grown as described in A.

Bacteria diluted to a 0D800 of 0.2 to 0.4 are then placed in a surface sterilized used for infecting disc sections from ginseng.

To surface sterilize the carrots, peel the carrots and sterilize them with a 10% solution of Clorox. Soak in the liquid for 20 minutes. Rinse the carrots with sterile water, cut into 5 mm pieces and soak in water. Place the base side up on top of the gar. Add 20 to 50 μl of bacteria to the top of the disc. Apply to the surface.

D. Transformation of sunflower Agrobacterium is grown as described in A.

Bacteria diluted to a 0D600 of 0.2 to 0.4 were then prepared as follows. used to infect the stems of infected sunflower plants.

Soak sunflower seeds in 10% captan for 1o, then 10% clorox Soak for 10 minutes and rinse with sterile water. Remove seed coat and add 0.7% ice water Germinate 5F3rIII in the dark on agar and then keep them at 28 °C and day and night. Transfer to a labyrinth incubator set for 2 hours. Let the seedlings grow for a week , followed by tip excision and bacterial infection of the cut stem surface.

E.) Transformation of madrids Agrobacterium is grown as described in A.

Bacteria diluted to a 0D600 of 0.2 to 0.4 were then prepared as follows. used to infect the stems of tomato seedlings.

Soak tomato seeds in 10% Clorox for 20 minutes and rinse with sterile water. seed were germinated for S days in the dark on 0.7% ice-water agar and then incubated at 23°C and daylight. and transferred to a labyrinth incubator set for 12 hours at night. Leave seedlings alive for one week The stem is allowed to lengthen, and then the tip is excised and the surface of the cut stem is infected with bacteria.

F. Transformation of cotton Grow Agrobacterium as described for humans.

Bacteria diluted to a 0D800 of 0.2 to 0.4 were then prepared as follows. used to infect cotyledons of treated cotton.

Soak the cotton seeds in ]0% Clorox for 20 minutes and rinse with sterile water. seeds Germinate in the dark on 0.7% ice water agar. The seedlings were allowed to grow for one week, and then the cotyledon surface Infect the surface with bacteria.

The superior inbred strain Funk 2717 of the Zea Mays plant was grown in a greenhouse until flowering; to self-pollinate. Immature panicles containing embryos approximately 2 to 2.5 mm long are removed from plants. Remove and sterilize for 20 minutes in a 10% Clorox solution. Remove the embryo from the grain Mycologically removed and with the hypocotyl facing down, 0.1 mg/12.4-D, 6% ( w/v) 0.24% (W/V) gel containing sucrose and 25mM L-proline. Place on 0MS medium (starting medium) solidified with Lurite (at 27°C in the dark for 2 weeks). After culturing at B5 medium containing 2.4-D and solidified with 0.24% (w/v) Gelrite Mborg et al., 1988). Cultivate callus on fresh medium every two weeks do. After a total of 8 weeks after placing the embryos on the starting medium, *another type of callus has developed its characteristics. Identified by characteristic morphology. This callus is further subcultured in the same medium. difference After another 2 months, the callus was treated with N containing 2 mg/12.4-D and solidified with Gelrite. 6 medium and serially subcultured.

The calli described in G. are subcultured for at least 6 months in total. for subculture The type of callus selected for As it separates, it is relatively non-stick, spherical and very brittle. big Culture media containing aggregates of poorly grown cells are not retained. There Maze Excellent Inbred 500mg of a special type of callus from Funk 2717 is added to 125ml of prongs. Place in 30 ml of N6 medium containing 2 mg/12.4-D in a lasco. rotary Culture I1 at 26 °C in the dark on a shaker (180 rpm, 2.5 cm travel distance) After one week, transfer the medium to fresh medium. The suspension is subcultivated again after a week in this way. nourish Inspect the culture at this time and keep those that are thick (no proliferated cells). Ru.

Discard the suspension culture containing aggregates with heavily proliferated cells. preferred organization are closely packed cells with characteristically smooth surfaces compared to those of normal cell aggregates. Consists of mitotic cell aggregates. The retained culture fluid appears in these small aggregates. have at least 50% of the cells.

This is the preferred configuration. These suspensions have a doubling time of less than one hour. It has a fast growth rate. Suspension culture solution is 0.5ml PVC (filling cell volume: pipette The cells are subcultured by transferring the set cell volume (in a set of cells) into 25 ml of fresh medium. . After 4 to 6 weeks of subculturing in this way, the culture solution! Weekly passage 2- to 3-fold increase per culture. 7596 or more cells is the desired morphology The culture solution is maintained by further subculturing. Fras showing its best form Strains are maintained by constantly selecting the co-content for subculturing. Pore size 68 Periodically passing through a 0 μm stainless steel sieve can enhance the dispersion of the culture medium. This is done in some cases, but not necessarily.

1 to 1.5 ml of PVC of the suspension culture prepared in H was added to 4% (w/v) cells. Lase R3 and KMC (8.65g/lKCl, 16.47g/lMgCl□・ 6H.

0.12.5g/]　CaC]* @2Hs 0.5g/IMES, pH5, 8) 10 to 1 filter-reduced mixture consisting of 1% (w/v) o-zyme in salt solution Keep warm in a 5m wide area. Digest at 30℃ for 8 minutes on a gently shaking table. Do this for 4 hours. The preparation is followed under an inverted microscope for protoplast release. release Collect the protoplasts produced as follows. Transfer the prepared solution to a 100 μm mesh. sieve and then filter through a 500 μm mesh sieve. Simulate protoplasts Wash on the tube with a volume of KMC salt solution equal to the initial volume of enzyme solution. P Place 10ml of rotoplast preparation into various disposable plastic centrifuge tubes. and 0.6M sucrose solution (0°1% (W/V) morpholinoethane sulfone buffered with acids (MBS and KOH) to pH 5.8) as the lower layer. did. Centrifuge the tube at 60 to 1100X for 0 min and collect at the interface. Collect whole protoplasts with a pipette and place into a new tube. proto The plast preparation was resuspended in 10 ml of fresh KMC salt solution and Centrifuge at 1100X for 5 minutes. Remove and discard the supernatant and add to the remaining drops. Gently resuspend the protoplasts and then add 10 ml of 18/14 strength KMC solution. 1 gradually. After centrifuging again for 5 min, the supernatant was removed again and Resuspend the protoplasts in 677 strength KMC solution. I counted some of the The cells are collected and pelleted again by centrifugation. Protoplasts, KM- 8p medium or 0.5M7nitol containing 6mM Mg C1g or form Resuspend at 10" per ml in other suitable medium for transformation.

a. All steps except heat shock are performed at room temperature (22-28°C). P At the final stage of rotoplast 1, 0.1% (w/v) M E S and resuspended in 0.5M mannitol containing 8mM Mg C1x.

The resistance of this suspension is determined by the Dialog Electrovolator. Mbeher, F-4000 D-4000 Dusseldorf) chamber. , and adjusted to 1.2 ohm using 800mM MgCl solution. Ru. The tube containing the sample was immersed in a 45°C water bath for 5 minutes at 45°C and then placed on ice. Infuse the protoplasts with thermosis by cooling them to room temperature. Roth Plant-selective hygromycin as described by Stein et al. (1987) 4g of linearized plasmid or chimeric gene construct containing resistance gene and calf breast 20 μg of glandular carrier DNA is added to a 0.25 ml aliquot of the above suspension. 0.5M Contains nitol! 24% (w/v) PEG in 30mM MgCl (MW8 000) solution is added to the protoplasts. Thoroughly mix the mixture. Mix slowly and keep warm for 10 minutes. Place the sample in an electrovolator into the chamber and pulse the sample three times at 10 second intervals (1500. Initial voltage of 1800.2800 or 2800Vcrn-', exponential function of 1 μsec target destruction time),.

Protoplasts are cultured as follows. Brake the sample in a 60 m betri dish at room temperature. Ting. After a further 5 to 15 minutes, 1.2% (w/v) G-Plaque aga Add 3 ml of KM-81) medium containing loin and 1 mg/l 2.4-D do. Mix the agarose and protoplasts thoroughly and allow the medium to gel. let

b. a is repeated with one or more of the following transformations: (1) The resistance of the protoplast preparation solution is adjusted to 0.5 to 0.7 ohm. .

(2) The PEG used has a molecular weight of 4000t.

(3) PEG is not added or 12% (w/v) PEG 1/2 volume is added.

(4) Pulses are applied at 3 second intervals.

(5) In a dish where protoplasts are placed on a plate cooled to a temperature of 16 °C. electroporation followed by blating.

(6) Place the protoplasts in a tube after electrovoresication, 67 7 strength KMC solution or W5 solution (!380mg/IKc1.18.375g /ICaClx・2Hz 0.9 g/INaCl, pH 6,0) 1 ．． KM medium 0 ml, then collected by centrifugation at 60 x g for 10 min, ．． Resuspend in 3 ml and brate as in a.

(7) Do not add calf thymus carrier DNA.

a, In the final step of step I, protoplasts were incubated with 12 to 30 mM M g C1. Resuspend in 0.5M mannitol solution. Heat shock at 45℃ for 5 minutes Proceed as described in . Part for transformation of protoplasts in centrifuge tubes Disperse the protoplasts in a volume of 0.3 ml per tube. Next Add the following substances for 10 minutes: DNA (as in J) and PEG solution. (MW6000, 40% (W/V); 0, IMCa(No)s and and 0.4M mannitol; adjust the final PEG concentration at pH 8 to 9) with KOH. It shall be 20%. Incubate a portion for 30 minutes with occasional rocking, then incubate the protoplasts. into a Petri dish (0.3 ml of initial protoplast suspension per 6 cm diameter). , and culture as described in J.

Repeat steps b and a and inject the protoplasts with 0.0% W5 solution 5 times at intervals of 2 to 3 minutes. After incubating for 30 minutes in the PEG solution by adding 8 ml of Ru. Centrifuge the protoplast suspension, remove the supernatant, and remove the protoplasts. culture as described in Example J, a.

c. Repeat a and b except the final PEG concentration is between 13 and 25% (w/v). Go back.

L. Regeneration of callus from protoplasts Plants containing protoplasts in agarose are placed in the dark at 26°C. 14 days later , colonies arise from protoplasts. 2 mg of agarose containing colonies/ 12. N6 medium 30 containing 4-D and solidified with 0.24% (w/v) Gelrite Transfer to the top of a 9 cm diameter vetri dish containing m1. This medium was named 2N6. Ru. The callus was further incubated in the dark at 26°C, and the callus pieces were incubated with fresh solid 2N6. Subculture on medium every two weeks.

Selection of transformed callus of M. zea mays Select transformed cells Add 100mg/l or 2oomg/l hygromycin B to 2NS medium for Repeat L except for the addition modification.

N. Regeneration of corn plants a, Calli were placed on 2N6 medium for maintenance and OH2 for initiation of regeneration. (NG medium without 2,4-D) and N61 medium (0.25 mg/l 2. 4-D and N6 medium containing xomg/l kinetin). OH2 and Callus growing on N61 medium was placed in the light (840 to 8400 lumens from a white fluorescent light). Grow in camphor light for 16 hours/day). Extended time on N61 medium Transfer callus growing on N61 medium to ON6 medium after 2 weeks as it is harmful. . Subculture the callus every two weeks, even if the callus is transferred again to a similar media formulation. do. Plantlets appear in about 4 to 8 weeks. The plantlets are at least 2 cm tall. Once reached, transfer to ON6 medium in GA7 containers. Roots will form in 2 to 4 weeks, Then, once the roots are sufficiently formed to support growth, place the plantlet in the soil in a beet pot. Transfer to soil and keep in the shade for the first 4 to 7 days. 2 to 8 to aid acclimatization I often place a clear plastic cup upside down over the plantlets. Often advantageous. Once the plants are established, treat them as normal corn plants and Maturing in a greenhouse. To obtain progeny, plants are self-pollinated or wild-type crossbreed with

b. 100 mg/l or 200 mg in the medium used to maintain callus. Repeat step a, except with the modification of adding /l hygromycin B.

Example 8 Genetics of transgenic plants obtained from Nidoransji Technik T8 seed line The following method for naming offspring type is used here: The first plant grown from culture is designated as T1 plant.

Plants resulting from self-pollination of natural flowers of T1 plants are designated as T2 plants. This is usually acquired a new genotype during meiosis. Similarly, T2 plants The seeds resulting from self-pollination of natural flowers (grown from 12 seeds) were named T8. Ru. The same applies below.

Allow the transgenic plants (T1) to mature. Self-pollinating flowers, and usually Collect the seed pods after drying. Collect seeds from individual plants and store them separately . For each seed loft, determine the number of Mendelian loci that give rise to kanamycin resistance traits. To do so, test by gene segregation analysis. 12 seeds contain 2% hypochlorite Surface sterilize with 0.02% (V/V) Tween 2G, then rinse with sterile water. . Approximately 150 seeds were seeded with 0.2XM containing 150μg/m1 kanamycin. S salt (Murashige and Skoog.

1962) on a filter paper saturated with Germination and cotyledon growth to approximately 5 mm Later, the ratio of normal green cotyledons (KAN-3) to bleached cotyledons (KAN-3) is determined.

Only T2 seed lots exhibiting a ratio of approximately 3:1 (Kango:Kan-s) Save for further analysis. This segregation ratio produces the kanamycin marker gene. evidence for a single Mendelian locus.

4 to 10 plants matured from each T2 seed lot (same conditions as above) and self-grown. Pollinate. Collection of 18 seeds, seed sterilization, and seed germination for 12 seeds Do the same as. 100% of seeds tested (n=150) had kan-r phenotype is homozygous for the trait (i.e. homozygous T3 seed lots exhibiting 2a plants) and preserved for phenotypic analysis.

Conventional breeding techniques are then used to obtain all genes in the same plant.

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BoRrman, J, EMBOJ, 6:2519-2523 (19g 7) Torres-3chuann, S., Godoy, J.Pint or-Toro, Icy Pbcnt MoL BioL 1 ■ j a49-7 57 (1992) Velten, J, Velten, L, )Liin, PL, 5che melon J,, EMBOJ, 3:2723-2730 (1([S) YamadLM, Plant Ce1l PhysioL 33:1-6 (1992) Wang, Y-C, etsL, Ph1nt MoL Bio L11:433-439 (X988)Weitf+g, L5chel LJ, Kahl, a, mual Rev, 0enet, 22:42 1-477 (1981P1) EP 164,575 EP434,616 FJ’46λ■6 WO8910437m US 4,945,050 OB　2,140.822 Sequence list Sequence number: 1 Sequence type: peptide Array length: 11 Ala e Thr Cys Gly Gin Val Ser Ser A la Leu sequence number = 2 Sequence type: peptide Array length: 28 Ala engineering 1a Sar Cys Gly Gin Val Ser Ser A la Lau Sar Pro Cygla Ser Tyr Ala Ar g Gly Asn Gly Ala Sequence number: 3 Sequence type: peptide Array length: 90 Ala Engineering 1a Thr Cys Gly Gin Val Ser Ser A la XAu Gly I’ro Cys 14AlaA1a Tyr Ala Lys Gly Ser Gly Thr Ser Pro Ser Ala Gly 28Cys Cys Sir Gly Val Lys Arg L eu Ala Gly L@u Ala Arg Ser -42ThrAla Asp Lys Gin Ala Thr Cys Arq Cys Leu Lys Se1: Val 56Ala Gly Any Tyr Asn Ala Gly Arg Ala Ala Gly Engineering 1e Pro Ser 7 0AXq CYII Gly Val Sar Val E’ro Tyr T hr engineering 1 @ Ser Ala Ser Val 84Aspcys Ser L ys engineering 1e) Iis 90 sequence number: 4 Sequence type: peptide Array length: 90 Ala engineering 1e Ser Cys Gly Gin Val Ser Ser A la Leu Ser Pro Cys 14k le Ser Dingyr Ala Arg Gly Asn Gly Ala Lys Pro Pro Ala Ala 28Cys Cys Ser Gly Val Lys Arg L eu Ala Gly Ala Ala Gin Ser 42Thr Ala Asp Lys Gin Ala Ala Cys Lys Cyglla Lys Ser Ala 56juaGly Gly I, eu Asn AL a Gly Lys Ala Ala Gly Engineering 1e Pro Ser 70 Me Ce Cys aly Val Sar Val Pro Tyr Ala xle Sar ALa Sar val 84Asp Cys Ssr Ly s Engineering Arg 90 Sequence number: 5 Sequence type: peptide Array length = 90 Aha Engineering Thr Cys Gly Gln’Val Sir Sar ALa Leu Gly Pro Cys x4Ale Ala Tyr Al a Lys Gly Ala Gly Val Asn Pro Ser Al a Gly 28Cys Cys Ser Gly Val Lys Arg ku Ala Gly Leu Ala Arg Ser 42? hr　ALa Asp Lys Gin Ala Thr Cys hxq Cys Lau Lys Ser Val 56Ala Gly Aly Tyr Juin Ala Gly Arg Ala Ala Gly X1* Pro Sar 70Aj-q Cya Gly Val Sir Val Pro ryr T hr Hls Ser Ala ser Val 84Asp Cys Sar 1Lys IIs Hls 9*Sequence number: 6 Sequence type: peptide Array length: 23 Ala engineering 1a Ser Cys Gly Gin Val Sar Ser A la Leu Ser Py: o Cys Engineering Ser Tyr Ala A rg Gly Ajn Asn Ala・α14 Tooni ]Δ LT26-+7: shi[tt+a-iJ■ CW21 0 CWlg Summary book The present invention provides novel anti-pathogen peptides, DNA sequences encoding the peptides, novel anti-pathogen peptides, a vector containing a DNA sequence, a vector containing a DNA sequence encoding the above peptide; compositions containing anti-pathogenic peptides and controlling plant pathogens The present invention relates to methods of using the peptides and compositions for the treatment of cancer. Anti-pathogen peptides may synthesize a method for producing a composition comprising the same and an anti-pathogen effective amount of the wrinkle peptide. Also included are methods of producing transgenic plants.

Submission of Translation of Written Amendment (Patent Law Article 184-7jlf Paragraph 1) 1993! 2nd day of the month □□□

Claims (44)

[Claims] 1. Amino acid sequence (SEQ ID NO: 3) [There is an array] anti-pathogen peptides containing. 2. Amino acid sequence (SEQ ID NO: 5) [There is an array] anti-pathogen peptides containing. 3. Amino acid sequence (SEQ ID NO: 4) [There is an array] anti-pathogen peptides containing. 4. Substantial sequence homology to the amino acid sequence according to any one of claims 1 to 3. A peptide containing an amino acid sequence with a specific property. 5. Isolation and purification of peptides from cell wall and membrane fractions of homogenized plant material The method for producing a peptide according to any one of claims 1 to 4, which comprises: . 6. 5. The method of claim 4, wherein the plant material is isolated from etiolated barley plants. 7. The peptide or fragment thereof according to any one of claims 1 to 4. Or a DNA sequence that encodes a part of it. 8. A vector comprising the DNA sequence according to claim 7. 9. Phospholipid transfer proteins or putative amylase/protease inhibitors A transgenic plant that expresses the encoding gene. 10. Transgens expressing genes encoding phospholipid transfer proteins plant. 11. The gene has an N-terminal sequence (a) [There is an array] or (b) [There is an array] or (c) [There is an array] encodes a peptide having at its N-terminus an amino acid sequence substantially homologous to The transgenic plant according to claim 9. 12. The gene encodes the peptide according to any one of claims 1 to 4. The transgenic plant according to claim 9. 13. A transgenic plant expressing a gene comprising the DNA sequence according to claim 7. thing. 14. Amino acid sequence below: (a) [There is an array] or (b) [There is an array] (c) [There is an array] (d) an N-terminal amino acid sequence substantially homologous to at least one of the above sequences; An anti-pathogen effective amount of a peptide containing at its N-terminus a hydrophobic region consisting of one of an anti-pathogen composition, together with a suitable carrier conventionally used in peptide formulations. 15. X4 is Arg (arginine), Lys (lysine) or His (histidium) and X5 represents Gly (glycine) or Ala (alanine) The composition according to claim 14. 16. Amino acid sequence (SEQ ID NO: 3) [There is an array] 15. The composition according to claim 14, which contains an anti-pathogen peptide containing as an active ingredient. 17. Amino acid sequence (SEQ ID NO: 5) [There is an array] 15. The composition according to claim 14, which contains an anti-pathogen peptide containing as an active ingredient. 18. Amino acid sequence (SEQ ID NO: 4) [There is an array] 15. The composition according to claim 14, which contains an anti-pathogen peptide containing as an active ingredient. 19. An anti-pathogen effective amount of the active ingredients together with suitable carriers customary for agricultural peptide formulations. The active ingredient consists of phospholipotransfer protein and lytic peptide. Antipathogen composition. 20. An anti-pathogen effective amount of the active ingredients together with suitable carriers customary for agricultural peptide formulations. wherein the active ingredient is the peptide according to any one of claims 1 to 4; An anti-pathogen composition comprising bacterial and lytic peptides. 21. 21. The method according to claim 19 or 20, wherein each component of the active ingredients is present in a synergistically effective amount. Composition. 22. 21. The composition according to claim 19 or 20, wherein the lytic peptide is thionin. 23. Applying an anti-pathogen effective amount of phospholipid transfer protein to the plant pathogen habitat A method of protecting plants from attack by phytopathogens. 24. An anti-pathogen effective amount of the peptide according to any one of claims 1 to 4 is inoculated. from attack of plant pathogens, consisting of application to objects or habitats of plant pathogens. How to protect plants. 25. Applying the anti-pathogen composition according to any one of claims 14 to 22 to a plant, or protect plants from attack by plant pathogens by applying to the habitat of the plant pathogen. How to protect. 26. Claim 28: The plant pathogen is a pathogenic bacteria and/or fungus. 25. The method according to any one of item 25. 27. Claim 23 consisting of prophylactically or therapeutically applying the anti-pathogen composition. 26. The method according to any one of items 25 to 25. 28. The peptide according to any one of claims 1 to 4 is used as an agricultural peptide preparation. homogeneously mixed with one or more suitable carriers and/or auxiliaries customary in A method for producing an anti-pathogen composition comprising: 29. Effective in the anti-pathogen effective composition according to any one of claims 14 to 22. A method using phospholipid transfer proteins as ingredients. 30. Effective in the anti-pathogen effective composition according to any one of claims 14 to 22. A method of using a peptide according to any one of claims 1 to 4 as a component. 31. The antipathogen effective composition according to claim 14 contains the following amino acid arrangement as an active ingredient. Column: (a) [There is an array] or (b) [There is an array] (c) [There is an array] (d) an N-terminal amino acid sequence substantially homologous to at least one of the above sequences; Using a peptide containing at its N-terminus a hydrophobic region preferably consisting of one of How to do it. 32. An anti-pathogen effective amount of the peptide according to any one of claims 1 to 4 is inoculated. plant pathogens, preferably A method of protecting plants from attack by pathogenic bacteria and/or fungi. 33. An anti-pathogen effective amount of the composition according to any one of claims 14 to 22. phytopathogens, preferably applied to plants or to phytopathogen habitats. is a method of protecting plants from attack by pathogenic bacteria and/or fungi. 34. Expressing anti-pathogen effective amounts of phospholipid transfer proteins in transgenic plants A method for controlling plant pathogens comprising causing them to appear. 35. Produces an anti-pathogen effective amount of the peptide according to any one of claims 1 to 4. A method for controlling plant pathogens comprising causing them to appear. 36. It consists of expressing an effective amount of an anti-pathogen active ingredient, and the active ingredient is Method for controlling plant pathogens consisting of follipotransfer proteins and lytic peptides . 37. It consists of expressing an anti-pathogen effective amount of an active ingredient, and the active ingredient is A plant comprising the peptide according to any one of items 1 to 4 and a lytic peptide. Methods of controlling pathogens. 38. A peptide produced by the method according to claim 5. 39. Following stages: a) Recombinant DNA encoding one or more phospholipid transfer proteins Create a transgenic plant containing the sequence, b) Injecting one or more phospholipid transfer proteins into the transgenic plant. synthesize A method for controlling plant pathogens consisting of. 40. Following stages: a) Any of claims 1 to 4 comprises one or more peptides according to claim 1. b) producing a transgenic plant containing a recombinant DNA sequence to be coded; Transducing one or more peptides according to any one of items 1 to 4 Synthesize in genic plants A method for controlling plant pathogens consisting of. 41. Following stages: a) A trans-transplant containing a recombinant DNA sequence encoding an effective amount of an anti-pathogen active ingredient. Generic plants are produced, and the active ingredient is according to any one of claims 1 to 4. b) Antipathogenic to transgenic plants. A body-effective amount of an active ingredient is synthesized, and the active ingredient is according to any one of claims 1 to 4. consisting of the peptide and lytic peptide described in A method for controlling plant pathogens consisting of. 42. Following stages: a) Recombinant DNA encoding one or more phospholipid transfer proteins Create a transgenic plant containing the sequence, b) Injecting one or more phospholipid transfer proteins into the transgenic plant. synthesize A method for controlling plant pathogens consisting of. 43.3) Encoding phospholipid transfer proteins and optionally lytic peptides b) producing a transgenic plant containing a recombinant DNA sequence containing the recombinant DNA sequence; Recombinant DNA encoding lipid transfer proteins and optionally lytic peptides Two or more transgenic plants containing the A sequence are created and conventionally claim for an anti-pathogen effective amount, comprising: crossing said plant using breeding techniques for Item 20. A method for producing a transgenic plant capable of synthesizing the active ingredient according to item 19. 44. a) a peptide according to any one of claims 1 to 4 and optionally Transgenic plants containing recombinant DNA sequences encoding lytic peptides are produce or b) a peptide according to any one of claims 1 to 4 and optionally a lytic peptide; Two or more transgenic sequences containing recombinant DNA sequences encoding peptides producing a genetic plant and hybridizing the plant using conventional breeding techniques; 36. A transducer capable of synthesizing an anti-pathogen effective amount of the active ingredient according to claim 35 consisting of How to create genetic plants.