RU2603058C1 - Peptide stellaria media l. starwort, having antifungal activity - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry, particularly to a peptide Sm-AMP3 possessing antifungal activity.

EFFECT: invention enables to efficiently protect plants against pathogenic fungi.

1 cl, 1 dwg, 1 tbl, 4 ex

Description

The invention relates to biochemistry, to biologically active peptides with antifungal activity, which can be used in biotechnology to create pathogen-resistant forms of crops and in the food industry for preserving food.

Pathogens and pests cause enormous damage to crops, reducing yields and worsening the quality of the final product. The most important measures to combat pathogens are breeding for resistance and the use of chemical plant protection products - fungicides and pesticides. The first approach is time-consuming and lengthy, the second, although effective, causes significant harm to the environment. New prospects for increasing plant resistance to pathogens have opened up with the development of genetic engineering, which allows the integration of individual genes that determine resistance from one species to another. The most tangible results were achieved when embedding the entomotoxin genes of the soil bacterium Bacillus thuringiensis, which made it possible to obtain plants resistant to a number of pests [de Maagd RA, Bosch D., Stiekma W. Bacillus thuringiensis toxin-mediated insect resistance in plants // Trends Plant Sci. - 1999. - Vol. 4. - P. 9-14].

Genetically modified plant varieties that are resistant to viruses have already been obtained and marketed. The first such plant was papaya resistant to the spotted virus, which was achieved by incorporating the envelope protein into the genome [Ferreira SA, Pitz KY, Manshardt R., Zee F., Fitch M., Gonsalves D. Virus coat protein transgenic papaya provides practical control of papaya ringspot virus in Hawaii // Plant Dis. - 2002. - Vol. 86 - P. 101-112]. A commercial genetically modified potato cultivar resistant to leaf curl virus and a Bt gene was commercialized in the 1990s. Resistance to the virus was provided by inhibiting the activity of the replicase gene [Delvas M., Ceriani M.F., Collavita M., Butzonich I., Norr N.E. Analysis of transgenic potato plants expressing potatao leaf-roll virus coat protein gene // Plant Physiol. - 1993. - Vol. 102 - P. 174-182].

However, genetically modified crops resistant to phytopathogenic fungi are still not presented on the market.

Despite the enormous opportunities that genetic engineering offers to increase the resistance of agricultural plants to diseases caused by pathogens, many questions remain unclear. In particular, the environmental consequences of embedding prokaryotic genes into the plant genome and the safety of such transgenic varieties for mammals [Vazquez-Padron RI, Moreno-Fierros L., Neri-Bazan L., de la Riva GA, Lopez-Revilla R. Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice // Life Sci. - 1999. - Vol. 64. - P. 1897-1912]. More promising and safe for use as transgenes are genes that determine the resistance of the plants themselves. It has been established that during the evolution of plants, they developed a complex multilevel system of protection against pathogens, in which compounds of various nature participate. The most important role among them belongs to antimicrobial peptides (AMPs), which provide resistance to a wide range of pathogens. Wild plants have increased resistance to biotic stress, largely due to the presence of highly active AMPs. The prospect of using AMP genes to increase plant resistance is determined by the fact that they can be directly integrated into the genome of plants sensitive to pathogens using genetic transformation methods [Carlini C.R., Grossi-de-Sa M.F. Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides // Toxicon - 2002. - Vol. 40. - P. 1515-1539].

The structure and antifungal properties of the claimed peptide are closest to the SmAMP1.1a peptide isolated previously from the seeds of Stellaria media (PDB ID: 2KUS A). The substance belongs to the family of cysteine-rich chitin-binding peptides, inhibits the growth of some pathogenic fungi in concentrations of 1.6-5.4 μM.

The invention solves the problem of expanding the range of peptides of plant origin with antifungal activity.

The problem is solved due to the structure of the new Sm-AMP3 peptide having the amino acid sequence of SEQ ID No. 1.

The inventive peptide exhibits pronounced antifungal activity against the following plant pathogen fungi: Fusarium solani, Aspergillus niger, Bipolaris sorokiniana, Botrytis cinerea, Alternaria alternata. To completely inhibit the growth of these fungi in vitro, micromolar concentrations of the peptide are necessary. The technical result of the invention is the high antimicrobial activity of the claimed peptide.

The Sm-AMP3 peptide consists of 35 amino acid residues. The charge at pH 7.0 is 2.0, and the isoelectric point is 10.43. The peptide can be obtained by chemical synthesis or biotechnologically.

The Sm-AMP3 peptide is obtained from the leaves of woodlice Stellaria media L., which belongs to the family Cloves Caryophyllaceae, the dicotyledonous class Dicotyledones, the angiosperms or flowering plants Magnoliophyta (Angiospermae).

The invention is illustrated by the following examples.

Example 1

Isolation of Sm-AMP3 Peptide

The stellate leaves are crushed in liquid nitrogen and extracted with 10 volumes of 10% acetic acid (w / v) with the addition of a mixture of proteinase inhibitors (Sigma-Aldrich, USA) (1 ml of solution per 30 g of plant material in 100 ml of buffer) at room temperature for 1 h with vigorous stirring. After centrifugation (10 min, 22000 g, 4 ° C), the precipitate is discarded, and the supernatant is concentrated 2 times on a rotary evaporator (Ika-Werke, Germany). Proteins and peptides are precipitated with chilled acetone in a ratio of 1: 7 (v / v) at 4 ° C overnight. After centrifugation (15 min, 22000 g, 4 ° C), the precipitate was collected and dried in air. The protein-peptide fraction is desalted by reverse phase high performance liquid chromatography (RP-HPLC) on a NewGuard C ₁₈ column (Applied Biosystems, USA) at an elution rate of 1 ml / min. The column is pre-equilibrated with solution A (0.1% aqueous trifluoroacetic acid). After applying the acid extract to the column, it is washed with solution A until the absorption value of the eluate becomes equal to the initial value. Proteins and peptides are stripped from the column with 60% acetonitrile in 0.1% TFA, evaporated in a vacuum concentrator to remove acetonitrile and freeze-dried.

, диаметр частиц 5 мкм; The Nest Group, Inc., США). Фракционирование проводят в линейном градиенте концентрации ацетонитрила: 5-40% раствора Б (80% CH₃CN в 0,1% ТФУ) за 60 мин при скорости элюции 1 мл/мин, а затем 40-60% Б за 15 мин. Детекцию проводят по оптическому поглощению при 214 нм (рис. 1). Пептид элюируется со временем удерживания 40 мин. В результате получают препарат искомого пептида с содержанием примесей менее 3%.The obtained dry residue from 10 g of the feedstock is dissolved in 2.5 ml of solution A and separated by RP-HPLC on a Vydac C ₁₈ column (4.6 × 250 mm, pore size 300

particle diameter of 5 microns; The Nest Group, Inc., USA). Fractionation is carried out in a linear gradient of acetonitrile concentration: 5-40% solution B (80% CH ₃ CN in 0.1% TFA) for 60 minutes at an elution rate of 1 ml / min, and then 40-60% B for 15 minutes. Detection is carried out by optical absorption at 214 nm (Fig. 1). The peptide elutes with a retention time of 40 minutes. The result is a preparation of the desired peptide with an impurity content of less than 3%.

Example 2

Establishment of the amino acid sequence of the Sm-AMP3 peptide

, диаметр частиц 5 мкм; The Nest Group, Inc., США), предварительно уравновешенной 0,1%-ной трифторуксусной кислотой. Продукты реакции и реагенты отделяют промывкой колонки этим же растворителем до достижения исходного оптического поглощения элюата, пептид элюируют в тех же условиях, которые описаны в примере 1 для очистки пептида Sm-AMP3.To determine the number of cysteine residues in the amino acid sequence of the peptide, disulfide bonds are restored and the formed thiol groups are alkylated. Reduction and alkylation are carried out according to a modified method [Thomsen J., Bayne S. Microscale alkylation with 4-vinylpyridine // J. Protein Chem. - 1988. - Vol. 7. - P. 295-296]. The peptide (1 nmol) was dissolved in 35 μl of a buffer containing 6 M guanidine hydrochloride and 2 mm EDTA in 0.5 M Tris-HCl buffer, pH 8.5), 2 μl of an aqueous solution containing 1 μmol 1.4- was added dithiothreitol, purged with nitrogen, stirred and incubated at 40 ° C for 4 hours. Then, 2 μl of a 50% solution of 4-vinylpyridine in 2-propanol are added to the mixture, stirred and incubated in the dark for 20 minutes at room temperature. 120 μl of 0.1% trifluoroacetic acid was added to the mixture, and the reduced and alkylated peptide were separated from the reaction products by reverse phase high performance chromatography on a Vydac C ₁₈ column (4.6 × 250 mm, pore size 300

particle diameter of 5 microns; The Nest Group, Inc., USA), pre-equilibrated with 0.1% trifluoroacetic acid. The reaction products and reagents are separated by washing the column with the same solvent until the initial optical absorption of the eluate is achieved, the peptide is eluted under the same conditions as described in Example 1 for the purification of the Sm-AMP3 peptide.

The amino acid sequence of the purified reduced and alkylated Sm-AMP3 peptide is determined by the Edman stepwise degradation method using a Procise 492 automated sequencer (Applied Biosystems, USA). As a result, the complete amino acid sequence of SEQ ID No. 1 of the Sm-AMP3 peptide, consisting of 35 amino acid residues, is established.

Example 3

Determination of the relative molecular weight and the number of free and disulfide-linked cysteine residues in the Sm-AMP3 peptide

The obtained amino acid sequence of SEQ ID No. 1, as well as the identity of the purified peptide, is confirmed by mass spectrometric analysis. Mass spectra were obtained on a MALDI time-of-flight mass spectrometer Ultraflex II TOF / TOF (Bruker Daltonik, Germany) with identification of positive ions in a reflex mode. The matrix used is a-cyano-4-hydroxycinnamic acid (CHCA) (15 mg / ml in 80% (v / v) acetonitrile containing 0.1% (v / v) trifluoroacetic acid). To calibrate the device using a standard mixture of peptides with a molecular weight range of 700-3500 Da (Sigma, USA).

The measured monoisotopic molecular weight of the natural Sm-AMP3 peptide is 3362.9 Da and within the accuracy of the MALDI mass spectrometer (0.1%) does not differ from the calculated (4431.66 Da). The molecular weight of the alkylated unreduced peptide Sm-AMP3 is 3363.0 Da, i.e. differs from the measured molecular weight of the natural peptide by 0.1 Da. Thus, the Sm-AMP3 peptide does not contain free sulfhydryl groups, and all 6 cysteine residues are involved in the formation of 3 disulfide bonds, the peptide does not contain any other chemical modifications.

Example 4

Biological properties of the Sm-AMP3 peptide

To determine the antifungal activity of the fractions obtained by separating acid-soluble proteins and peptides of woodlouse, as well as the purified Sm-AMP3 peptide, the following fungal cultures are used: Fusarium solani, Aspergillus niger, Bipolaris sorokiniana, Botrytis cinerea, Alternaria alternata.

To study the activity, the isolated fractions and the purified Sm-AMP3 peptide were frozen in liquid nitrogen, freeze-dried, re-dissolved in water and again dried. Solids are redissolved in 50 μl of water. The concentration is determined by the absorption spectrum in the UV region. Antifungal activity is determined by the inhibition of spore germination in a liquid nutrient medium (potato dextrose solution) in 96-well plates. A series of twofold dilutions of the test substances in water is prepared. The test sample and the spore suspension of the fungus are placed in the wells. The total sample volume is 100 μl. As a control, a well is used in which, instead of the solution of the test sample, distilled water is added. The plates are incubated for 48 hours and the inhibition of spore germination is evaluated by the optical density of the suspension at 620 nm.

Determination of the peptide concentrations necessary for 50% inhibition of fungal spore germination (IC ₅₀ ) is carried out by the double dilution method. The results for the Sm-AMP3 peptide are given in table. one.

Claims (1)

Peptide Sm-AMP3 having antifungal activity, having the amino acid sequence of SEQ ID No. 1.

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