Applied Biochemistry and Microbiology最新文献

A technology for sugar beet genotyping has been developed (Beta vulgaris) at ten microsatellite loci: FDSB1001, FDSB1033, SB04, SB09, SB15, Unigene16898, Unigene17623B, Unigene17923, Unigene26753, and Unigene27833. The technology can be used for efficient, accurate, and rapid identification of sugar beet lines and hybrids. The proposed approach includes multiplex PCR for all loci in one test tube followed by electrophoretic analysis of the obtained DNA fragments in one capillary of a genetic analyzer. One of the key elements of the technology, determining the accuracy and reproducibility of the analysis, is the use at the electrophoresis stage of an additional length standard, an allelic ladder consisting of DNA fragments of the analyzed microsatellite loci. The advantages of the proposed technology include the possibility of standardization and automation of the method in a 96-well plate format, which allows for the establishment of a mass analysis process. As a result of this study, the DNA profiles of six modern sugar beet hybrids (Azimut, Visit, Korvet, Rubin, Fregat, and Uspekh) and their component lines were obtained for the first time. The developed technology can be used effectively for genetic identification of sugar beet lines, assessment of the homogeneity of plant material, and quality control of hybridization at all stages of the breeding process and can become a reliable laboratory tool for supporting industrial seed production of this crop.

Environmental DNA (eDNA) is genetic material obtained directly from samples of various natural substrates, rather than directly from a living organism. The use of eDNA simplifies monitoring of secretive and low-abundance species that are difficult to detect using classical environmental survey methods. Understanding the capabilities and limitations of eDNA-based methods requires information about the influence of various factors on the rate of degradation of nucleic acids in the environment. We studied the rate of DNA degradation at a temperature of 14°C in water from Svirskaya Bay of Lake Ladoga with pH 7.3 in the absence of light. For this purpose, highly specific primers and a TaqMan probe were developed and tested for the D-loop fragment of mitochondrial DNA of the domestic chicken (Gallus gallus domesticus). The choice of this particular genetic material as exogenous DNA was due to the minimal risk of contamination of natural water in the sampling area. At the start of the experiment, chicken DNA (galDNA) was added to the water samples to a concentration of 50 μg/L and detected by real-time PCR after 6 h and 1, 2, 3, 8, and 14 days. Using the developed protocol, we identified target D-loop sequences in natural water samples during the first three days of the experiment, with trace amounts of galDNA detected up to eight days.

The production of the most studied, active and widespread groups of brassinosteroids by salt-tolerant bacteria Priestia megaterium Ср-1, Rhodococcus jostii CA-6 and Pseudomonas koreensis FP2/1 isolated from saline soil samples was studied. It was shown that the studied bacterial strains are capable of producing steroidal phytohormones of the brassinolide, 24-epibrassinolide and 28-homobrassinolide groups, as well as brassinosteroid B-ketones, B-lactones and 6-deoxo derivatives. This circumstance may be the cause of the growth-stimulating activity of these bacteria and their effect on plant adaptation to stress factors.

The effects of exogenous abscisic acid (ABA) on early germination processes in seeds of two cereal crops, wheat and triticale (a hybrid of wheat and rye), are investigated. A clear relationship was established between growth processes and both the concentration of ABA and the imbibition stage at which ABA was applied. At moderate to high concentrations (10^–6–10^–4 M), ABA acted as an inhibitor of physiological processes. Early stages of seed germination, specifically those involving physical swelling and radicle emergence, were found to be highly sensitive to ABA. Furthermore, a high concentration of exogenous ABA (10^–4 M) inhibited the mobilization of protein reserves in the wheat embryo, which may contribute to the overall inhibition of germination.

Upon immunochemical determination of low-molecular-weight compounds capable of binding only to one antibody molecule (haptens), competitive interactions of the hapten in the sample and the hapten-carrier conjugate (usually, a hapten-protein conjugate) with the antibodies are conducted. The characteristics of such analytical systems largely depend on the properties of the conjugates used. However, the relationships between the conjugate composition and the achieved limit of detection (LOD) are described fragmentarily and are not systematized. In this study, a panel of nine conjugates of bisphenol A, a relevant toxic environmental pollutant, with soybean trypsin inhibitor (STI) and bovine serum albumin is considered. The composition of the conjugates synthesized by the carbodiimide-succinimide method was determined using fluorescamine. Concentration dependences of binding between antibodies and conjugates immobilized in microplates and competitive interaction with free bisphenol A were studied. The enzyme-linked immunoassay (ELISA) implemented using various conjugates was characterized by LODs and working ranges. It was demonstrated that the minimum LOD of 1.9 ng/mL was achieved using the bisphenol A–STI conjugate with a 5 : 1 composition. The developed ELISA was successfully tested for the detection of bisphenol A in natural and drinking water.

A highly efficient expression system for recombinant Escherichia coli type II L-asparaginase (EC 3.5.1.1) has been developed based on a synthetic gene optimized for the energy of secondary structure hairpins formation in the 5'-region of mRNA. A producer strain, E. coli BL21[DE3]/pET28a-AsnSYN was created, providing an accumulation of up to 291 ± 9 mg/L of enzymatically active protein (44.5 ± 2.6 mg/(L AU)) when cultivated in stirred flasks, which is 50% higher than the control strain with a natural gene at an induction time of 3 hours. Optimization of the codon composition of the gene made it possible to increase the energy of secondary mRNA structure formation in the 5'-region from –70 to –47 kcal/mol, which, as we assume, contributed to improved translation efficiency. For the developed producer strain, asparaginase that meets pharmacopoeial purity requirements can be obtained with a total yield of ≥25% and a specific activity of >250 IU/mg. The preparation contains no visible impurities according to electrophoresis data and less than 3% multimeric forms according to size exclusion chromatography data. The results obtained demonstrate the promise of using synthetic genes with optimized DNA structure for the industrial production of therapeutic enzymes.

In this work, the ability of endophytic bacteria Bacillus subtilis (Cohn.) (strains 26D and Ttl2) to suppress the reproduction of potato virus Y (PVY) and potato virus X (PVX) on tomato plants (Solanum lycopersicum L.) and to stimulate the growth of infected plants by regulating the redox balance was demonstrated. The bacterial strains B. subtilis 26D and B. subtilis Ttl2 reduced the titer of PVY and PVX in tomato plants, restored their growth to the control values, which was accompanied by a decrease in the disease symptoms and severity. PVY and PVX disrupted the redox balance of plants for their development. However, the treatment with B. subtilis 26D and B. subtilis Ttl2 strains regulated the generation of hydrogen peroxide by changing the activity of catalase and positively affected the activity of peroxidases in tomato plants infected with PVY or PVX. This allowed us to suggest the possibility of using these strains as a basis for creating biopreparations for the protection of tomato plants from viral diseases.

Using directly engineered derivatives of previously constructed succinate-producing Escherichia coli strain SUC1.0 (pMW119-kgd) (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, P_Lglk, P_tacgalP, ∆aceBAK, ∆glcB, ∆sdhAB, pMW119-kgd) the feasibility of gamma-aminobutyric acid biosynthesis from glucose by this bacterium resulting from a partial reversal of GABA-shunt was demonstrated. The formation of succinate semialdehyde from 2-ketoglutarate was ensured in the strain resulting from the expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase gene. Conversion of succinate semialdehyde to succinic acid was prevented by the inactivation of cellular NAD⁺- and NADPH⁺-dependent succinate semialdehyde dehydrogenases. Formation of the target substance by the action of native 4-aminobutyrate aminotransferase was achieved upon the inactivation of glutamate decarboxylases A and B. Enhancement of 4-aminobutyrate aminotransferase gene expression led to an increase in the molar yield of gamma-aminobutyric acid from glucose demonstrated by the strain synthesizing the target product through the partially reversed GABA-shunt from ~11 to ~25%.

The process of bioleaching of metallurgical slag containing nickel and copper was studied at 40, 45, and 50°C. The microbial communities grown at 40 and 45°C that included Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans bacteria were used in the experiments. The microbial community, which was cultivated at 50°C, included the representatives of S. thermosulfidooxidans and Acidiplasma sp. It was demonstrated that dissolution of the solid phase, oxidation of ferrous iron by microorganisms were the largest at 45°C. At the same time, the majority of copper (95.5–100%) and nickel (92.3–100%) passed into the solution at the first day of bioleaching. The study of the kinetics of chemical leaching of non-ferrous metals over 4 h demonstrated that in the presence of 5 g/L Fe³⁺, the extraction of nickel and copper was 93.0 and 94.3%, respectively, while in the absence of Fe³⁺, 75.0% of nickel and 77.8% of copper passed into the solution.

The work is devoted to the experimental study of Arthrospira platensis culture growth in a two-stage luminostat. A new method of flow culture is presented, which allows increasing the efficiency of light energy assimilation, as well as controlling the content of photosynthetic pigments in algal biomass. At the first stage, the growth of A. platensis culture at step 1 of luminostat in the batch mode was investigated. Due to high surface and spatial irradiance, there is a prolonged exponential phase on the growth curve. The maximum specific growth rate of the culture was 0.96 1/day and the chlorophyll a and C-phycocyanin content was low: 1.37 and 2.3%, respectively. The light energy passing through the first stage varied from 2.23 to 33.24% depending on the maintained density of the quasi-continuous culture. The reduction in light flux affected the maximum productivity of A. platensis at step 2 of luminostat, which was 0.08 g/(L day). In the second experiment, cultivation was carried out in a two-stage luminostat regime, when the nutrient medium sequentially flowed through both steps. It was shown that at the second stage, due to a decrease in irradiance, there was photoadaptation of A. platensis cells consisting in a slight increase in the proportion of chlorophyll a and a significant increase in C-phycocyanin: 13.6%. The pigment content was directly influenced by the uniformity of cell irradiation, being only 8% for the second stage, which led to a decrease in productivity and an increase in the content of C-phycocyanin. The findings allow us to recommend the two-stage luminostat for solving the problems of optimization in order to obtain microalgal biomass with desired pigment composition.