Inhibitor of andis virus of potato mottling
SUBSTANCE: invention relates to the use of the concentrate of the culture liquid of the strain Trichoderma harzianum Rifai, deposited in the Russian National Collection of Industrial Microorganisms under the number of RNCIM F-180 as an inhibitor of Andis virus of potato mottling.
EFFECT: invention enables to reduce losses of potato from the plant infection with the Andis mottling virus.
The invention relates to biochemistry and may find application in agricultural biotechnology, Microbiology and plant breeding.
A well-known inhibitor of the herpes virus simple 1-th type (1), a known inhibitor of human immunodeficiency virus (2). As inhibitor was used metabolite strain Trichoderma harzianum Rifai - homogeneous enzyme L-lysine α-oxidase.
In connection with the importation into Russia of a large number of imported material of potato, there is a risk of infection of healthy plants of the Andean potato virus craptastic potatoes. Also a great risk of spreading infected potato associated with an increase in the international exchange of seed and genetic material in the form of tubers and in vitro culture and true seeds (3, 4).
Andean virus craptastic potatoes (Andean potato mottle virus) is widely distributed in the highlands of the Andes in Chile, Ecuador and Peru, and Brazil. It mainly affects the potatoes, however, can be found on the eggplant and some other solanaceous crops (3, 5).
Description inhibitors Andes virus craptastic potatoes (Andean potato mottle virus) not found.
The technical result of the invention is to reduce losses from infection of potato plants in the Andes virus craptastic potatoes.
The technical result is achieved by the fact that the proposed application is concentrate the culture fluid of strain Trichoderma harzianum Rifai, deposited in Russian national collection of industrial microorganisms under the number F-180, as an inhibitor in the Andes virus craptastic potatoes.
The strain is known (6), deposited in Russian national collection of industrial microorganisms under the number VKPM F-180 (Moscow, Road 1-y passage, 1. Institute of genetics and breeding, 1987).
The cultivation conditions of a strain of Trichoderma harzianum Rifai.
Fermentation of the strain was carried out on the equipment Experienced process plant IBPM RAS. Gscreen (Pushchino).
Used the fermenter type BIOR-01 produced by BLS Bureau, city of Kirishi, volume 100 l with a fill factor of 0.6. The fermenter equipped with a magnetic stirrer, filters air, temperature and pH.
Nutrient medium was prepared directly in the apparatus. For this purpose, the apparatus was filled with 60 l of tap water, contributed 1% wheat bran, stimulant-0,1 %, 1.3% of ammonium sulfate, the pH value of 5.8-6.0 installed a 10% solution of Hcl. Wheat bran and stimulant pre-soaked in 10 litres of water for 4 hours, were sterilized in an autoclave for 1 hour at t° +125°C. Then prepared the fermenter was seeded with seed material from the flasks. Sowing dose of at least 5%. Cultivation was carried out at a temperature of +26°C, air flow throughout the fermentation 30 litres per minute, the speed is Esaki 200 rpm. The pH value in the process of growth was adjusted to 6.5. Duration of cultivation was 94-98 hours, the final pH from 5.3 to 7.5.
At the end of the fermentation culture liquid was sent to the site pre-treatment, where the fungal mycelium was separated by filtration under vacuum on the suction filter. The weight of the obtained biomass was 3.5 kg Received native solution was subjected to additional separation in the separator of the type RSD at 9000 rpm for one hour at a temperature of +2 to +4°C. Then the native solution in a volume of 55 l, obtained after separation of the biomass was concentrated to 1.5 l (concentrate).
Ribonucleic acid (RNA) in the Andes virus craptastic potato was extracted from positive control (firm DSMZ, Germany). The samples were ground in a porcelain mortar until a homogeneous state with the addition of lytic buffer from the set of "Sample NC", company OOO Agravante". From pre-prepared samples were isolated RNA using sets of "Standard NC", company OOO Agravante", according to the Protocol from a set.
For the production of reverse transcription (FROM) in a test tube was added the following mixture for FROM: FROM-buffer AMV company Promega (250 mM Tris-HCl, 250 mM KCl, 50 mM MgCl22.5 mM spermidine, 50 mM DTT) and 5 μl of the mixture dNTP company CJSC Dealt LTD (da, dt, DG, DC - 200 µm each), and 2.5 μl, Rnasin - 40 units (1 μl), revertase AMV firm "romega" (10 U/μl) - 3 μl of water to a total volume of 20 μl of the RNA sample was 5 μl. The reverse transcription reaction (the formation of double-stranded DNA by matrix-stranded RNA) occurred at t° +42°C for 1 hour, at t° +95°C - 5 minutes.
At the stage of reverse transcription to isolated and purified RNA virus, Andes virus craptastic potato was added to the concentrate of the culture fluid in different dilutions (from 5 to 0.025 U/ml). Next to amplification reaction was prepared with the following mixture: 10x MagMIX - 2025 closed joint stock company "Dialout-LTD - 2,5 μl of the sample, the primers of the Armu river direct and inverse 2 μl (10 ρmol/μl) to the sample, cDNA and 5 μl of water to 25 μl of mineral oil. As a result, the complimentary deoxyribonucleic acid (cDNA). To obtain the reaction products (amplicons) were heated mixture as follows: one cycle at t° +96°C for 15 minutes, 30 cycles at t° +96°C for 30 seconds, at t° +65°C for 30 seconds and at t° +72°C for 30 seconds; one cycle at t° +72°C for 10 minutes. Storage was carried out at t° +4°C. amplicons (repeatedly increased the number of copies of the study area cDNA) was made in a 1.5% agarose gel, after which he received electrophoregram and evaluate the results.
After carrying out reverse transcription to samples selected cDNA was added to the enzyme at concentrations of 1 U/ml of 2.5 U/ml and 5 U/ml Was also taken control sample cDNA, which was not added enzyme. On electrophoregram the presence of dark bands in the sample testified to the preservation of a virus, no stripes meant his destruction. At a concentration of 1 U/ml or more cDNA of the virus is destroyed, with no formation of the dark stripes.
After carrying out reverse transcription to samples selected cDNA was added to the enzyme at different concentrations (1, 0,1, 0,05, of 0.025 U/ml). Was also taken control cDNA sample without added enzyme. On electrophoregram the presence of dark bands in the sample testified to the preservation of a virus, no stripes meant his destruction. At a concentration of more than 1 U/ml cDNA of the virus was destroyed, this was not observed the formation of the dark stripes. At a higher dilution of the enzyme (0,1, 0,05, of 0.025 U/ml) viral cDNA is saved and there is a dark band.
Sources of information
1. Inhibitor of herpes simple 1-St type. RF patent № 2022012, 1994. S.B. Alekseev, birch CT, Andjaparidze OG and others.
2. Inhibitor of human immunodeficiency virus. RF patent № 2022011, 1994. S.B. Alekseev, Weight B.C., Smirnov I.P., and others.
3. ERRO. Phytosanitary procedures PM 3/21 (2). Post-entry quarantine for potato // Bulletin OEPP/EPPO Bulletin, 2004, vol.34, p.443-454.
4. Fribourg, C.E.; Jones, R.A.C.; Koenig, R. (1979) Andean potato mottle virus. CMI / AAB Descriptons of Plant Viruses No.203. Association of Applied Biologists, Wellesboume, UK.
5. Salazar, L.F.; Harrison, B.D. Particle properties and strains of Andean potato mottle virus. Journal of General Virology, 1978, 39, 171-178.
6. Smirnov I.P., birch CT, Chekunova LN. Strain Trichoderma harzianum Rifai producer L - Lisino-α-oxidase. The copyright certificate. 1991, No. 1044043.
The concentration of the culture fluid of strain Trichoderma harzianum Rifai, deposited in Russian national collection of industrial microorganisms under the number F-180, as an inhibitor in the Andes virus craptastic potatoes.
SUBSTANCE: group of inventions relates to biotechnology and can be applied in creation of analytic methods with application of peroxidases. Method includes preparation of substrate mixture, introduction of peroxidases in substrate mixture with the following registration of intensity of formed luminescence. Substrate mixture includes the following components, given in final concentrations: buffer solution 10-125 mM, luminol 0.05-8 mM, hydrogen peroxide 0.05-8 mM, 4-aminopyridines 0.1-10 mM, N-carboxyphenothiazine, or N-(carboxymethyl)phenothiazine, or N-(2-carboxyethyl)phenothiazine 0.1-10 mM. And pH of buffer solution constitutes 7.9-9.0.
EFFECT: invention ensures obtaining of high intensity of chemiluminescence and, accordingly, high sensitivity of peroxidase activity determination.
4 cl, 8 ex
SUBSTANCE: invention relates to biotechnology and discloses isolated polynucleotides which encode Δ8 desaturase. The invention also relates to Δ8 desaturases themselves, which encode isolated polynucleotides, expression vectors containing isolated polynucleotides, host cells containing expression vectors and methods of producing Δ8 desaturase and polyunsaturated fatty acids selected from a group consisting of dihomo-gamma-linolenic acid (DGLA), ω3-eicosatetraenoic acid (ω3-ETA) and any combinations thereof.
EFFECT: invention widens the range of Δ8 desaturases used to produce polyunsaturated fatty acids.
12 cl, 12 dwg, 14 tbl, 6 ex
SUBSTANCE: invention relates to field of biochemistry, in particular to genetic construction for expression of threonine-insensitive aspartate kinase in plant, where construction contains promoter, specific for phase of aging, functionally connected with coding sequence, which codes polypeptide, possessing activity threonine-insensitive aspartate kinase, and in which specific to aging promoter is selected from group, consisting of SAG12, SAG13, SAG101, SAG21 and SAG18, or their functional variants or fragments. Described are vector, plant cell, transgenic plant, material for plant reproduction, collected leaves, smoking tobacco, which contain claimed genetic construction. Also described is method of obtaining transgenic plant, which possesses ability to accumulate threonine in fading leaves in larger amount than in leaves of corresponding plant of wild type, and method of increasing the level of threonine in leaves of aging plant to the level of threonine, higher than the content of threonine in leaves of plant of wild type without impairing plant adaptation with application of said genetic construction.
EFFECT: invention makes it possible to obtain transgenic plant, which possesses ability to accumulate threonine in fading leaves in larger amount than in leaves of plant of wild type.
22 cl, 16 dwg, 2 tbl, 4 ex
SUBSTANCE: invention is the truncated mutant luciferase MLM4 of Metridia longa with the size of -16 kDa with improved properties for use as a genetically encoded bioluminescent reporter for visualisation of molecular processes in living cells. The amino acid substitutions I69L, N74E, K125V, W139F are introduced in the sequence of the truncated luciferase MLM4 of Metridia longa with the size of -16 kDa.
EFFECT: invention enables to provide the luciferase with improved thermal stability and the spectrum shift to long-wave region, which determines an overall increase in sensitivity of the bioluminescent reporter when used in living cells and organisms.
4 dwg, 1 tbl, 3 ex
SUBSTANCE: invention relates to the field of biotechnology and can be used for enantioselective enzymatic restoration of keto compound of general formula (I):
where R represents a protecting group for the functional amino groups, and X is -Cl, -CN, -OH, Br, F. The polypeptides which have oxyreductive activity, depending on the amino acid sequence restore the said compound to the corresponding R,S- and S,S-hydroxy compounds.
EFFECT: use of the invention enables to reduce the ability to form the by-products in restoration of keto compound of the formula.
6 cl, 5 ex
SUBSTANCE: versions of delta-12-oleatedesaturase (FAD2) proteins are presented, where one version has an amino-acid substitution in the position corresponding to the position 108 for aspartic acid, and the other one - substitutions in positions corresponding to positions 108 for aspartic acid and 118 for phenyl alanine, in the protein FAD2 of wild type with a sequence SEQ ID NO:4 or SEQ ID NO:8, given in the description. The following are described: nucleic acids, which code the specified proteins; vectors of expression containing the specified nucleic acids; master cells containing the specified vectors. Fragments are proposed, which contain at least 20 nucleotides of the extracted molecule of nucleic acid, containing the specified substitutions, for use as a primer, a probe and/or a marker. A plant is proposed, which creates a high profile of oleic acids in oil of seeds transformed by nucleic acid with a sequence SEQ ID NO:1, or SEQ ID NO:1 and SEQ ID NO:5.
EFFECT: invention makes it possible to increase content of oleic acid in seeds of oil-bearing plants.
19 cl, 2 tbl, 1 dwg, 3 ex
SUBSTANCE: method provides mixing a suspension of the microorganism with the substrate (10% solution of glucose) and a hydrogen acceptor, which is used as a methylene blue colorant followed by measuring the concentration of methylene blue during the enzymatic reaction. According to the rate of change of the concentration of methylene blue in the linear phase of the enzymatic reaction the dehydrogenase activity is determined.
EFFECT: invention enables to reduce time of determining the dehydrogenase activity of microorganisms and to improve the quality of its determination.
SUBSTANCE: tick-borne encephalitis virus is inhibited with the use of L-lysin-α-oxidase produced by the strain of Trichoderma harzianum Rifai, Russian National Collection of Industrial Microorganisms (VKPM) F-180.
EFFECT: invention enables inhibiting viral activity of tick-borne encephalitis in cells in vivo.
SUBSTANCE: what is presented is a method for preparing the substance L-lysine-alpha-oxydase (LO) with the use of the Trichoderma cf. aureoviride Rifai BKMF-4268D producer strain. It is followed by fermentation on a medium containing nitrogen, phosphate sources and wheat bran. It is followed by recovery and purification of the enzyme in the following sequence of the procedures: culture fluid dialysis, adsorbent treatment, impurity sedimentation in 20%-saturated ammonium, hydrophobic and ion-exchange chromatography. It is followed by preparing L-lysine-alpha-oxydase of molecular weight 115-116 kDa, isoelectric point 4.25, optimum pH - 7.4.
EFFECT: higher yield of LO.
5 tbl, 5 ex
SUBSTANCE: what is presented is a recovered polynucleotide for expression of delta-5-desaturase containing a nucleotide sequence: 1) coding a polypeptide of delta-5-desaturase with the polypeptide having at least 80% identity SEQ ID NO:2; 2) coding the polypeptide of delta-5-desaturase with the nucleotide sequence having at least 90% identity SEQ ID NO:1 ot SEQ ID NO:3; 3) coding the polypeptide of delta-5-desaturase with the nucleotide sequence being shock-hybridised with the nucleotide sequence presented in SEQ ID NO:1 or SEQ ID NO:3; 4) a complement of the nucleotide sequence 1)-3). There are described a recombinant DNA structure, a host cell, an oilseed transgenic plant, a transgenic seed containing said polynucleotide. The following methods are presented: host cell transformation, expression of long-chain polyunsaturated fatty acids in a plant cell, production of at least one polyunsaturated fatty acid in a plant with the use of said polypeptide. Usability of said transgenic seeds for producing human foodstuff, animal feed, oil and lecithin is described.
EFFECT: invention enables producing a changed profile of polyunsaturated fatty acids as compared with a profile of polyunsaturated fatty acids of a non-transformed plant.
24 cl, 11 tbl, 22 dwg, 20 ex
SUBSTANCE: nutrient medium for growing filamentous fungi-dermatophytes from clinical material comprises glucose, bacteriological agar, meat peptone, casein hydrolyzate, yeast extract, sodium chloride, sodium carbonate, L-cysteine, thioglycolic acid and distilled water in a predetermined ratio of components.
EFFECT: invention enables to reduce time of growing filamentous fungi-dermatophytes from clinical material.
1 tbl, 2 dwg, 4 ex
SUBSTANCE: invention relates to biotechnology. The method involves treating the sensitive layer of a biosensor with solution of a common fraction of protease of hepatopancreas of king crab in a buffer comprising tris-HCl 50 mM, CaCl2 3 mM, NaCl 100 mM, pH 8.0 at solution temperature of 35-40°C. The process is carried out in multiple successive steps with a solution of a solution of a common fraction of protease of hepatopancreas of king crab in said solvent at temperature of 35-40°C followed by exposure. The sensitive layer of the biosensor is successively washed with dodecyl sulphate dissolved in the same buffer in concentration of 0.1% and then with water at each step. The treatment process is carried out three times.
EFFECT: invention reduces the duration of the process.
SUBSTANCE: method of obtaining an antiviral preparation is carried out by preparation of an inoculation mycelium of basidiomycete Enokitake Flammulina velutipes (Curtis) Singer, preparation of a liquid nutritional medium, which contains water, vegetable oil and molasses as a carbon source, as a nitrogen source - corn flour, as mineral salts - potassium dihydrophosphate and magnesium sulphate, its sterilisation, inoculation of the sterile nutritional medium with the prepared inoculation mycelium, cultivation of basidiomycete in it in an aerobic conditions; the obtained submerged culture is divided into basidiomycete biomass and a culture liquid, from which a clot is separated by addition to the latter of ethyl alcohol, which is pressed, dried and crushed with obtaining the antiviral preparation under specified conditions.
EFFECT: method makes it possible to simplify technological process, increase the output of the antiviral preparation, possessing higher activity.
2 cl, 1 tbl, 4 ex
SUBSTANCE: strain of fungus Aspergillus oryzae Amy T-52-3-21 produces maltogenic α-amylase, and it is deposited in the All-Russian Collection of Microorganisms Institute of Biochemistry and Physiology of Microorganisms n.a. GK Scriabin RAS under the number F-4476D. The strain is made on the basis of strain Aspergillus oryzae of All-Russian Collection of Microorganisms F-3927D using the genetic engineering methods. Activity of α-amylase at 120 h of growth of the strain is 600-640 units/ml.
EFFECT: higher level of activity of maltogenic α-amylase.
1 tbl, 2 ex
SUBSTANCE: invention relates to biotechnology. Claimed is strain of Fusarium sambucinum, deposited in VKPM collection under number F-1161. Claimed strain is producent of protein food biomass.
EFFECT: invention makes it possible to accumulate biomass with high protein content with higher quantity of valuable unsaturated fatty acids, complete in composition.
2 tbl, 3 ex
SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining food fungal biomass with high protein content. Multicycle deep cultivation of Fusarium sambucinum All-Russian collection of industrial microorganisms F-1161 on liquid nutritional medium, containing sources of carbon, nitrogen, mineral salts, separation and drying of wet fungus biomass are carried out. Cultivation is performed at pH from 3.5 to 7.0 under conditions of air aeration from 0.5 to 2.0 l/l/min. Temperature mode in each cycle of fermentation is supported from the beginning of the cycle to the point of switch at the level from 26 to 30°C, and further to the end of the cycle at the level from 22 to 25°C. Point of switch is determined by accumulation of biomass to concentration from 45 to 60% from maximally achievable in fermentation apparatus, or point of switch is determined by concentration of dissolved oxygen by its reduction to the value from 20 to 40% of saturation ( calculated per atmospheric air pressure).
EFFECT: invention makes it possible to obtain the largest accumulation of biomass with high protein content with specified quantity of nucleic acids.
SUBSTANCE: mutant strain is obtained by impact on Glarea lozoyensis ATCC 20957 strain by nitrosoguanidine and it is deposited in CGMCC with number CGMCC 2933.
EFFECT: fungi strain has stable genetic and productive properties, produces low quantity of impurities during fermentation and is acceptable for commercial production of antibiotic.
6 cl, 2 dwg, 2 tbl, 3 ex
SUBSTANCE: detection method of microfungi Coccidioides posadasii 36 S and Coccidioides immitis C-5 in vitro involves pre-growth of culture in mycelial phase, preparation of a suspension corresponding to 5 units of activity of a standard opacity sample, possibility of spherules formation and detection of spherules filled with endospores. Culture in mycelial phase is grown during 3 days. Possibility of spherules formation is provided by infection of the one-day culture of cells of murine splenocytes, which is obtained on RPMI-1640, and further cultivation during 5 days at the temperature of 37°C, with content of CO2 in atmosphere of 5%. In order to detect spherules in the form of round double-outline formations filled with endospores, a sample is taken, deactivated with formalin and investigated by means of a light microscopy method.
EFFECT: invention allows simplifying the method and reducing the investigation period.
SUBSTANCE: production method of keratinase provides for directed adaptation of strain Penicillium citrinum PC-54-91"ВИЛАР" by three-time subcultivation on Czapek agar medium containing 2% of hair keratin as a carbon source. Cultivation under deep conditions on Czapek medium containing 10% of hair keratin and 0.5% of saccharose during 6 days. Separation of biomass from culture fluid with further extraction of target product by two-stage chromatographic cleaning involving gel filtration on TSK-Gel TOYOPEARL HW-40 and affine chromatography on protein A to sepharose CL-4B with further lyophilisation.
EFFECT: invention allows increasing ferment yield, obtaining keratinase preparation decomposing the hair α-keratin, and improving cleaning degree of ferment preparation.
2 tbl, 5 ex
SUBSTANCE: proposed method provides for preparation of inoculating mycelium of basidiomycete, which has been chosen from the following groups: Flammulina velutipes (Curtis) Singer and/or Hericium erinaceus (Bull.) Pers. Preparation of culture medium containing pulverised cold-pressed sunflower cake, soya flour, potassium dihydrogen phosphate, magnesium sulphate and water in the specified ratios. Inoculation of the obtained culture medium with the inoculating mycelium of basidiomycete, cultivation of basidiomycete with further separation of mycelium biomass and extraction of an antitumour agent from the biomass.
EFFECT: invention allows improving an environmental situation due to utilisation of production wastes of food industry.
3 cl, 1 tbl, 4 ex
SUBSTANCE: strain of unicellular algae Dunaliella salina IPPAS D-295-producer of biologically active substances having antioxidant activity was deposited in the culture collection of microalgae of the Institute of Plant Physiology n.a. K.A.Timiryazev RAS (IPP RAS (IPPAS)) under registration number IPPAS D-295 and can be used to produce biologically active substances having antioxidant activity and antagonistic activity against opportunistic pathogenic bacteria.
EFFECT: invention enables to increases the yield of antioxidant substances.
3 tbl, 2 ex