4/2017

Author

page: 3-12 pp
DOI: 10.11134/btp.4.2017.1
Rahimzhanova A.O., Kubash Zh.A., Ramankulov Y.M., Manabaeva Sh.A.
National Center for Biotechnology
Kоrgalzhyn road, 13/5, Astana, 010000, Kazakhstan
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Abstract

Genetic modification of cotton is given special attention due to its high economic value. The control of weeds that have a negative impact on its yield is one of the main problems in agriculture. As a result, the genetic engineering has become an unavoidable tool for reducing the negative impact of herbicides, causing significant damage to field crops. The aim of this work was to study the regeneration processes, and also to obtain herbicide resistant cotton plants by using Agrobacterium-mediated transformation. A recombinant construct pBPAT-GFP containing the genes phosphinothricin acetyltransferase PAT and GFP was created. The regeneration features of Kazakhstani varieties of cotton such as Mahtaaral-4005, Turkestan-1, Atakent-2010 in tissue culture were studied and incompetence to shoot formation was revealed, however, a single case of the formation of primordia from the callus was observed with the combination of hormones 2,4-D and zeatin in concentrations of 0,5 mg/l and 0.2 mg/l, as well as 2iP and NAA in concentrations of 5.0 mg/l and 0.1 mg/l. An effective method for regenerating shoots from stem apical meristems of transformed plants has been developed. Intensive shoot formation of two-day apical meristems of cotton with the content of two phytohormones with cytokine activity of BAP and kinetin at a concentration of 2.0 mg/l was revealed.

Keywords: cotton (G. hirsutum), growth regulators, regeneration, Agrobacterium-mediated transformation.

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Author

page: 12-16 pp
DOI: 10.11134/btp.4.2017.2
Turdiyev Т.Т.1, Frolov S.N.1, Madiyeva G.А.1, Rymkhanova N.K.2, Kovalchuk I.Yu.1
1Institute of plant biology and biotechnology,
Almaty, Kazakhstan, Timiryazev str., 45, 050040
2Al-Farabi Kazakh National University,
Almaty, Kazakhstan, 71,  Al- Farabi ave., 050040
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Abstract

The southern and south-eastern regions of Kazakhstan have exceptionally favorable conditions for the cultivation of pear. However, in recent years, the areas occupied with this culture are gradually decreasing. Many foreign and domestic cultivars, valuable hybrids and local traditional cultivars of folk breeding carrying genes of high productivity and adaptability have been lost in the field genebanks (orchards and nurseries). It is necessary to study, collect and preserve valuable genotypes that have important economic and biological characteristics to restore and prevent further loss of the gene pool.

Conservation of plant genetic resources in the field is associated with significant financial costs for the care of plantations, as well as the risk of loss of cultivars due to disease, pests and adverse environmental factors. The most promising and effective approach to solve this problem is the cryopreservation of plant germplasm in liquid nitrogen.

Studies on optimization of cryopreservation protocols for pear meristematic tissues were carried out based on vitrification method with 0.3 M sucrose. It has been established that the viability of meristematic tissues after cryopreservation depends on the duration of hardening, the method of thawing, the type of cryoprotectant and genotype. Optimum duration of hardening by variable temperatures (8 h, 22°C, light 5.9 lx, then 16 h, –1°C, dark) is 3-4 weeks, and the best cryoprotectant in preparing apical meristems for cryopreservation is PVS2. An effective way for viability recovery is thawing in water at a temperature of +45°C, and then at +25°C followed by planting on a nutrient medium.

Keywords: pear, biotechnology, gene pool, genetic resources, meristematic tissues, cryopreservation, cryobank.

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Author

page: 17-24 pp
DOI: 10.11134/btp.4.2017.3
Gritsenko D.A., Deryabina N.D., Galiakparov N.N.
Institute of plant biology and biotechnology,
Timiryazev str., 45, Almaty, 050040, Kazakhstan
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Abstract

Grapevine virus A belongs to the Vitivirus genus. Grapevine virus A is involved in the etiology of Kober stem grooving, a disease of the grapevine rugose wood complex.  The grapevine virus A genome (7.4 kb) consists of five open reading frames. The ORF2 function is not currently investigated. We attempted to develop a vector, based on grapevine virus A by cloning the gene encoding the capsid protein of apple chlorotic leaf spot virus in ORF2. Modification of grapevine virus A genome has not affected amplification or movement of virus through the plant. Symptoms of infection were detected 6-7 day after agroinfiltration. Nevertheless, immunoblotting has not detected the capsid protein of the capsid protein of apple chlorotic leaf spot virus either in the infiltrated leaves or in the upper leaves on the 15th day after agroinfiltration. It may be explained by the weak subgenomic ORF2 promoter. In addition, the genetic stability of modified grapevine virus A after K gene insertion was confirmed.

Key words: Grapevine virus A, ORF2, capsid protein, movement protein, apple chlorotic leaf spot virus.

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Author

page: 25-29 pp
DOI: 10.11134/btp.4.2017.4
Kirillov S.O., Khassenov B.B., Silayev D.V.
National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
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Abstract

In this work, recombinant alkaline phosphatase (phoB) from Bacillus licheniformis was successfully expressed in Escherichia coli, purified and biochemically characterized. Gene coding alkaline phosphatase was amplified from genomic DNA of B. licheniformis and cloned into expression vector pET-28c (+). Using the recombinant vector, a BL21 (DE3)/pAlPh strain-producer was obtained with over expression of the gene. Optimal cultivation parameters for producing recombinant alkaline phosphatase have been determined; 10 mg of protein was purified from 1 liter of culture. The activity of the recombinant alkaline phosphatase is 100 U/mg at standard conditions. Biochemical characteristics of recombinant alkaline phosphatase showed that enzyme has maximum activity at pH=10.0 and temperature +60°C. The kinetics of p-nitrophenyl phosphate hydrolysis have been studied, the Michaelis constant Km was 0.91±0.13 mM and the limiting value of the maximal rate of the enzymatic reaction Vmax was 21.4±1.19 mM. Experiments were carried out to determine the dependence of the enzyme activity on various divalent metals.

Keywords: alkaline phosphatase, strain-producer, biochemical characteristics, enzymatic activity, purification, expression, kinetic parameters.

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Author

page: 30-36 pp
DOI: 10.11134/btp.4.2017.5
Kamalova D.K.1, Kuybagarov M.A.1, Abeev A.B.2, Shvedyuk V.B.1, Balykbaev K.O.2, Kiyanbekova L.S.2, Aushakhmetova Z.T.2, Shevtsov A.B.1
1National Center for Biotechnology,
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
2National Center of Expertise,
Zheltoksan str., 46,  Astana, 010000, Kazakhstan
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Abstract

Enterovirus infections are widespread throughout the world. The clinical manifestation of enterovirus infection can lead to serious and fatal complications. Express PCR diagnosis and genotyping allows controlling the epidemiological situation and predicting the severity of the infection. In Kazakhstan, there are no locally produced PCR test kits. In addition there is no regular monitoring of circulating genotypes of enteroviruses. This study was aimed to develop a PCR protocol for the enterovirus detection based on the amplification of the 5' untranslated region (5' NTR) and genotyping by sequencing of enteroviruses circulating in Kazakhstan in 2016 - 2017. In this study 74 cDNA samples were used. The results obtained with developed protocol were well correlated (97.3%) with the results of the commercial PCR testing kit. The advantage of the developed protocol is genotyping of enteroviruses by sequencing of the amplified DNA fragment. Fifty nine samples were genotyped, of which 54.7% belong to enterovirus B, 35.9% are classified as enterovirus A and 1.56% are classified as enterovirus C. Ninety-three percent of meningitis-associated genotypes were identified as enterovirus B, and clustered with E-9, E-18, CV-B5 and E-30. Samples of DNA that isolated from patients with enterovirus infection were clustered with the CV-A6 genotype.

Key words: enterovirus, PCR, genotyping, 5 'NTR region, serous meningitis, intestinal infection

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Author

page: 37-40 pp
DOI: 10.11134/btp.4.2017.6
Zakarya K.D.1, Sarmurzina Z.S.1, Dospaeva R.T.1, Bissenova G.N.1, Shulgau Z.T.2, Gulyaev A.E.2, Krivoruchko T.N.2, Zhetpisbaev B.B.3
1Republic Collection of Microorganisms,
Valikhanov street, 13/1, Astana, 010000, Kazakhstan
2National Center for Biotechnology,
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
3National Center of Neurosurgery,
Avenue Turan, 34/1, Astana, 010000, Kazakhstan
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Abstract

The article describes experimental results of preclinical tests of the combined biopreparation “Microfit” intended for prevention and correction of intestinal microflora. The preparation consists of lactic acid bacteria of the genus Lactobacillus, an extract of balsamic poplar and tagansorbent. Pre-clinical study of the safety of the biopreparation “Microfit” on the state of internal (absolute and relative mass) organs of laboratory animals was conducted. According to the results, it was established that the biopreparation “Microfit” with the course of intragastric rat administration to rats at the conventional therapeutic dose (30 mg/kg) and the dose 10 times higher than the conventional therapeutic dose (300 mg/kg) had not led to a general toxic and destructive effect on the internal organs of animals. The safety of the biopreparation “Microfit” after intragastric administration and good tolerance of animals was confirmed. The results allow approving safety of the tested biological product.

Keywords: research, biopreparation, toxicity, conditional therapeutic dose, laboratory animals, internal organs.

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