RU2352580C1 - Peptide with antifungal activity - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: from satin flower (Stellaria media (L.) Vill.) a peptide is obtained, exhibiting antifungal activity against phytopathogens Alternaria consortiale, Fusarium culmorum, Helminthosporium sativum (syn. Bipolaris sorokiniana, Drechslera sorokiniana) and Thielatiopsis basicola.

EFFECT: wider assortment of peptides obtained from plants, with antifungal activity.

3 dwg, 1 tbl, 4 ex

Description

The invention relates to biochemistry, namely to biologically active peptides with antifungal action, which may find application in biotechnology.

The damage to world agriculture caused by pathogens of cultivated plants (fungi, bacteria, viruses and viroids) and pests (two main causes of damage) is estimated at trillion rubles annually. Losses often reach 45% of the crop. Modern methods used to reduce losses mainly come down to the use of synthetic pesticides, many of which are environmentally aggressive substances. The use of chemical plant protection products poses a significant environmental hazard; biological plant protection products are safer. The introduction of a new generation of insecticides allowed to improve the situation only by 5-10% [Oerke E.S., Dehne H.W., Schönbeck F., Weber A. Crop Production and Crop Protection: Estimated Losses in Major Food and Cash Crops. - 1994. - Elsevier, Amsterdam].

The use of genetic engineering methods for plant transformation opens up new possibilities for creating crops that are widely resistant to pathogens, pests, and abiotic stress (drought, soil salinity, etc.). For the first time, work on producing resistant cultivars using recombinant DNA technologies was performed about 20 years ago [Andrews R.E., Faust R.M., Wabiko H., Raymond K.C., Bulla L.A. The biotechnology of Bacillus thuringiensis. // Crit. Rev. Biotech - 1987. - Vol.6. - P.163-232. Vaeck M., Reynaerts A., Höfte H., Jansens S., De Beuckeleer M., Dean C., Zabeau M., Van Montagu M., Leemans J. Transgenic plants protected from insect attack. // Nature - 1987. - Vol. 328. - P.33-37]. Using the gene of the insecticidal protein of the bacterium Bacillus thuringiensis, transgenic tobacco plants resistant to the parasitic larvae of the Manducta sexta butterfly were obtained. Since then, at least 10 entomotoxin genes from Bacillus thuringiensis have been transferred and expressed in at least 26 plant species that have become resistant to certain pests [Schuler T.N., Poppy GM, Kerry BR, Denholm I. Insect-resistant transgenic plants. // Trends Biotech. - 1998 .-- Vol.16. - P.168-175]. A significant part of the world crop today falls on the share of Bt-transformed products (information taken from the official website of the US Department of Agriculture: www.aphis.usda.gov). Nevertheless, the issue of the safety of Bt-containing products for humans has not yet been resolved; the ecological consequences of the global spread of transgenic plants containing the genes of the entomotoxins Bacillus thuringiensis [Vazquez-Padron RI, Moreno-Fierros L., Neri-Bazan L. are not entirely clear. , 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]. In addition, a visible violation of the interspecific barrier receives a negative response in modern society. There are practically no other genetically modified varieties with increased resistance on the market. Moreover, the engineering of disease resistance has proven to be more challenging than increasing insect resistance.

According to modern concepts, the most promising approaches to obtaining resistant varieties are those that are aimed at enhancing the plant's own protective properties. Compounds produced by plants to protect against pathogens include the so-called phytoalexins and phytoantisipins - substances of various chemical nature, as well as some proteins and peptides. It is promising to obtain resistant varieties with altered expression of their own protective genes or transfer genes from one plant to another, for example, from wild to cultivated, because it is known that during selection and selection, agricultural plants, acquiring some useful traits, lost others. Of greatest interest are polypeptide compounds with protective properties, due to the possibility of their direct use to obtain transgenic plants [Carlini C.R., Grossi-de-Sā 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 most similar to the Ar-AMP peptide from the seeds of amaranth Amaranthus retroflexus [Lipkin A., Anisimova V., Nikonorova A., Babakov A., Krause E., Bienert M., Grishin E., Egorov T An antimicrobial peptide Ar-AMP from amaranth (Amaranthus retroflexus L.) seeds. // Phytochemistry. - 2005 .-- Vol.66. - P.2426-2431]. The substance belongs to an extensive family of cysteine-rich chitin-binding peptides that inhibits the growth of certain pathogenic fungi in concentrations of 3.5-30 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-AMP-1.1a peptide having the following amino acid sequence:

H ₂ N-Ser ^l -Gly ² -Pro ^c -Asn ⁴ -Gly ⁵ -Gln ⁶ -Cys ⁷ -Gly ⁸ -Pro ⁹ -Gly ¹⁰ -Trp ^ll -Gly ^l2 -Gly ¹³ -Cys ^l4 -Arg ^l5 -Gly ^l6 -Gly ^l7 -Leu ^l8 -Cys ^l9 -Cys ²⁰ -Ser ²¹ -Gln ²² -Tyr ²³ -Gly ²⁴ -Tyr ²⁵ -Cys ²⁶ -Gly ²⁷ -Ser ²⁸ -Gly ²⁹ -Pro ³⁰ -Lys ³¹ -Tyr ³² - Cys ³³ -Ala ³⁴ -His ³⁵ -OH

The inventive peptide exhibits pronounced antifungal activity against the following plant pathogen fungi: Alternaria consortiale, Fusarium culmorum, Helminthosporium sativum (syn. Bipolaris sorokiniana, Drechslera sorokiniana), Thielatiopsis basicola. 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 antifungal activity of the claimed peptide.

The Sm-AMP-1.1a peptide consists of 35 amino acid residues and can be obtained by chemical synthesis or biotechnological.

The Sm-AMP-1.1a peptide is obtained from a natural source - the seeds of a weed plant of medium stellate, or woodlice Stellaria media (L.) Vill., Which belongs to the clove family Caryophyllaceae, the class of dicotyledons Dicotyledones (Magnoliopsida), the department of angiosperms or flowering plants Magnoliophyta ( Angiospermae).

The invention is illustrated by the following examples.

Example 1

Isolation of Sm-AMP-1.1a Peptide

Starlet seeds are crushed in a coffee mill and extraction is carried out with 10 volumes of 10% (v / v) acetic acid at room temperature and stirring for 1 h. The suspension is centrifuged at 22000 g for 15 min at room temperature, the precipitate is discarded. The supernatant was further desalted by reverse phase high performance liquid chromatography on an Aquapore RP-300 C ₈ column (10 × 30 mm, pore size 300 Å, particle diameter 7.5 μm; Applied Biosystems, USA). The following solutions are used: A - 0.1% (v / v) trifluoroacetic acid in water, B - 80% (v / v) acetonitrile in 0.1% trifluoroacetic acid. The column is washed with 5 volumes of solution B, followed by equilibration with 5 volumes of solution A with an elution rate of 1.5 ml / min. Detection is carried out by optical absorption of the eluate at 214 nm. After applying the acid extract to the column, it is washed with solution A until the absorption level of the eluate is close to the original. Peptides and proteins are desorbed with solution B, the eluate is evaporated on a water-jet pump to remove acetonitrile, then lyophilized.

The resulting dry residue from 10 g of feedstock was dissolved in 4 ml of solution B (10 mM Tris-HCl, pH 7.2) and separated by affinity chromatography on a 25 × 40 mm column packed with Heparin Sepharose 6 Fast Flow media (GE Healthcare , USA) and previously equilibrated with buffer B. After applying the sample and washing out the non-absorbed substances, the peptides and proteins are first eluted with 100 mM NaCl in buffer B (fraction I) and then with 500 mM NaCl in buffer B (fraction II) at a speed of 1 ml / min The detection is carried out by optical absorption of the eluate at 280 nm (figure 1). The obtained fractions are desalted and dried as described above, and their biological activity is tested.

Fraction I was dissolved in 1 ml of solution G (5% (v / v) acetonitrile in 0.05% (v / v) trifluoroacetic acid) and separated by gel filtration on a 2.5 × 90 cm column filled with HiPrep Sephacryl S-100 HR vehicle (GE Healthcare, USA) and pre-equilibrated solution G. Separation is carried out at an elution rate of 1 ml / min and room temperature. The detection is carried out by optical absorption at 214 nm (figure 2). Fractions of 10 ml were collected, dried, and their biological activity was tested.

Fractions with an elution time of 240-280 min (figure 2), for which antifungal activity was detected, are combined and separated using reverse-phase high-performance liquid chromatography on a Vydac Ci ₁₈ column (4.6 × 250 mm, pore size 300 Å, diameter 5 μm particles; The Nest Group, Inc., USA). Fractionation is carried out in a linear gradient of acetonitrile: 5-40% solution B for 60 minutes at an elution rate of 0.7 ml / min and a temperature of 40 ° C. The detection is carried out by optical absorption at 214 nm (figure 3), test the obtained fractions. The Sm-AMP-1.1a peptide elutes with a retention time of 41 minutes. An additional purification step is carried out under the same conditions. The result is the preparation of the desired peptide with an impurity content of less than 3%, which is confirmed by chromatography on a Luna C ₁₈ analytical column (1 × 150 mm, pore size 100 Å, particle diameter 3 μm, Phenomenex, USA) in a linear gradient of acetonitrile concentration: 5- 40% solution B in 60 min at an elution rate of 50 μl / min, detection is carried out by optical absorption at 210 nm.

Example 2

Establishment of the amino acid sequence of the Sm-AMP-1.1a peptide

To unambiguously identify cysteine residues in the amino acid sequence of the peptide, disulfide bonds are restored and free thiol groups are alkylated. Recovery and alkylation is 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) is dissolved in 40 μl of buffer (6 M guanidine hydrochloride and 2 mm EDTA in 0.5 M Tris-HCl, pH 8.5), 2 μl of an aqueous solution containing 1 μmol of 1,4-dithiothreitol is added, thoroughly purged with nitrogen, mix, precipitate drops and leave at 40 ° С for 4 hours. Then add 2 μl of a 50% solution of 4-vinylpyridine in 2-propanol, mix the tube carefully, and precipitate drops. The alkylation reaction is carried out in the dark at room temperature for 15 minutes. Then the mixture was diluted 4 times with 0.1% trifluoroacetic acid, applied to a Vydac C ₁₈ column (4.6 × 250 mm, pore size 300 Å, particle diameter 5 μm; The Nest Group, Inc., USA), previously balanced with 4% acetonitrile in 0.1% trifluoroacetic acid. Reagents and reaction by-products are separated by washing the column with the same solution until the eluate absorption level is close to the initial one, then chromatography is carried out under the conditions described in Example 1 for the purification of Sm-AMP-1.1a peptide.

The N-terminal amino acid sequence of the purified reduced and alkylated Sm-AMP-1.1a peptide was determined by Edman stepwise degradation on a Precise 492 automated sequencer (Applied Biosystems, USA). As a result, the complete amino acid sequence of Sm-AMP-1.1a is established, consisting of 35 amino acid residues:

H ₂ N-Ser ^l -Gly ² -Pro ³ -Asn ⁴ -Gly ⁵ -Gln ⁶ -Cys ⁷ -Gly ⁸ -Pro ⁹ -Gly ¹⁰ -Trp ^ll -Gly ^l2 -Gly ^l3 -Cys ^l4 -Arg ^l5 -Gly ^l6 -Gly ^l7 -Leu ¹⁸ -Cys ^l9 -Cys ²⁰ -Ser ²¹ -Gln ²² -Tyr ²³ -Gly ²⁴ -Tyr ²⁵ -Cys ²⁶ -Gly ²⁷ -Ser ²⁸ -Gly ²⁹ -Pro ³⁰ -Lys ³¹ -Tyr ³² - Cys ³³ -Ala ³⁴ -His ³⁵

Example 3

Determination of the relative molecular weight and the number of free and disulfide-linked cysteine residues in the Sm-AMP-1.1a peptide

The obtained amino acid sequence, as well as the identity of the purified peptide, is confirmed by mass spectrometric analysis. Mass spectra were obtained on an ultraflex II TOF / TOF MALDI time-of-flight mass spectrometer (Bruker Daltonik, Germany), with identification of positive ions in a reflex mode. Dihydrobenzoic acid (10 mg / ml) in 50% (v / v) acetonitrile containing 0.1% (v / v) trifluoroacetic acid is used as a matrix. 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-AMP-1.1a peptide is 3460.20 Da. The calculated mass of the peptide with free thiol groups for cysteine residues is 3466.36 Da, i.e. differs from measured by 6.16 Yes. The calculated mass of the peptide under the condition of the formation of three disulfide bonds is 3460.31 Da, i.e. differs from the measured by 0.11 Yes. Thus, the Sm-AMP-1.1a peptide does not contain free thiol groups, and all 6 cysteine residues are involved in the formation of 3 intramolecular disulfide bonds, the peptide does not contain any additional chemical modifications.

Example 4

Biological properties of the peptide Sm-AMP-1.1a

To determine the antifungal activity of the fractions obtained during the separation of the acid extract of stellate seeds and the purified peptide Sm-AMP-1.1a, cultures of the following pathogenic fungi from the class Deuteromycetes are used: Alternaria consortiale, Fusarium culmorum, Helminthosporium sativum (syn. Bipolaris sorokiniana, Drechslera sorokiniana, Drechslera sorokiniana, Drechslera ), Thielatiopsis basicola.

To study the activity, the separated fractions are frozen in liquid nitrogen and freeze-dried, redissolved in water, and dried again. Finally, solids are dissolved in 50 μl of sterilized water. The concentration is determined by the absorption spectrum in the UV range. Activity is studied by radial diffusion on a nutrient medium with agar. Mushrooms are grown for two days on Petri dishes with medium prepared from a mixture of cereal flakes Nestle (20 g / l) and agar, with temperature control (26 ° C). Then a fungus block 3 mm in diameter is cut out and placed in the center of the Petri dish (9 cm in diameter) with medium so that the mycelium is pressed against the surface of the cup, left for 48 hours at the same temperature. The test samples (50 μl) are placed in wells with a diameter and depth of 3 mm, cut in agar at a distance of 3 cm from the center of the cup. 4 wells are cut out on each dish, one of which serves as a control - 50 μl of clean sterilized water is placed here. Petri dishes are incubated for two days in a thermostat, after which the inhibitory (fungistatic) activity of the samples on the growth of the fungus (by the width of the zone of inhibition in comparison with the control) and their effect on morphological changes in the development of mycelium are evaluated.

The determination of the minimum concentrations of peptides necessary to inhibit the growth of fungi (minimum effective concentrations) is carried out by the double dilution method. The results for the Sm-AMP-1.1a peptide are shown in the table.

Table
Antifungal properties of the peptide Sm-AMP-1.1a Mushroom The minimum effective concentration, μm Alternaria consortiale 0.7 Fusarium culmorum 2.1 Helminthosporium sativum 0.7 Thielatiopsis basicola 5,6

Claims (1)

A peptide having antifungal activity against phytopathogens Alternaria consortiale, Fusarium culmorum, Helminthosporium sativum (syn. Bipolaris sorokiniana, Drechslera sorokiniana), Thielatiopsis basicola, having the following amino acid sequence:
H ₂ N-Ser ¹ -Gly ² -Pro ³ -Asn ⁴ -Gly ⁵ -Gln ⁶ -Cys ⁷ -Gly ⁸ -Pro ⁹ -Gly ¹⁰ -Trp ¹¹ -Gly ¹² -Gly ¹³ -Cys ¹⁴ -Arg ¹⁵ -Gly ¹⁶ -Gly ¹⁷ -Leu ¹⁸ -Cys ¹⁹ -Cys ²⁰ -Ser ²¹ -Gln ²² -Tyr ²³ -Gly ²⁴ -Tyr ²⁵ -Cys ²⁶ -Gly ²⁷ -Ser ²⁸ -Gly ²⁹ -Pro ³⁰ -Lys ³¹ -Tyr ³² - Cys ³³ -Ala ³⁴ -His ³⁵ -OH.

Images