2/2017

Author

page: - pp
DOI: 10.11134/btp.2.2017.1
Zholdybayeva E.V.
National Center for Biotechnology
13/5, Korgalzhyn road, Astana,010000, Kazakhstan
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Abstract

Multi-drug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) area major problem in Kazakhstan and, hence,new research initiatives and treatment protocols are urgently required.World-wide, diagnosis of TB is largely reliant on smear microscopy; however,this method has low sensitivity, which can also be further compromised in an endemic HIV setting. Moreover, smear microscopydoes not provide information on drug susceptibility or the genotype of the causative agent. At present, drug susceptibility is mainly tested using culture-based methods.However, the methodology is time-consuming and requires specialized infrastructure, and thus,in many countries,is restricted to large centers and does not always include testing for second-line drug resistance.An alternative approach is the TB-SPRINT assay, which reflects the idea of an “all-in-one” assay that identifies the causative agentand also identifieswith high confidence any mutations conferring resistance. The TB-SPRINT assaycan act as a surveillance tool to monitor the epidemiology of disease in communities and countries. An improved tuberculosis diagnostic assay and improved understanding of risk factors for spread of drug-resistant TB will benefit local populations and will indirectly be very beneficial to the economy of Kazakhstan.

Key words: Tuberculosis, MDR-TB, XDR-TB, strains, genotyping, SNP, mutation, TB-SPRINT

 

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Author

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DOI: 10.11134/btp.2.2017.2
Ryabushkina N.A., Pozharskiy A.S., Omasheva M.Y.
Institute of Plant Biology and Biotechnology,
Timiryazev str., 45, Almaty,050040, Kazakhstan
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Abstract

Horticultural perennial crops are cultivated on one-eighth of global agricultural areas and are significant contributors to world food production. Fruits and berries from such crops are recognized as essential for good nutrition and prevention of numerous diseases. However, developing cultivars that meet modern economic and environmental demands require many breeding cycles and a considerable period. Modern varieties are developed to show high quality and yield, and resistance to pathogens and pests. The recent developments in high-throughput DNA sequencing technologies have been exploited to identify the associations between genes and/or genomic intervals controlling important traits and phenotype. These technologies also enable the development of molecular markers for assisted selection and breeding, the introgression of useful traits from wild relatives such as disease resistance, fruit quality, and rootstock characteristics. Genomic methods provide a valuable approach for the selection of traits of interest in seeds or seedlings and for the improvement of breeding efficiency through marker-assisted selection (MAS) and genome-wide association studies (GWAS). The accurate and cost-effective characterization of large collections of diverse wild germplasms supports such breeding initiatives. Grapevines and apples are among the most economically important of perennial crops and are affected by a large number of pathogens. The monogenic nature of many resistance genes has allowed the identification of homologs in wild relatives and enables the development of molecular markers linked to resistance loci. Thus, genomic approaches in combination with traditional breeding methods offer a promising prospect for improving perennial crops.

Keywords: perennials, crop wild relatives, molecular markers, next generation sequencing, genomics-assisted breeding, disease resistance, grapevine, apple.

 

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Author

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DOI: 10.11134/btp.2.2017.3
DeryabinaN.D.1*, GritsenkoD.A.1,2, GaliakparovN.N.1
1Institute of plant biology and biotechnology
Almaty, Kazakhstan, Timiryazevstreet, 45, 050040
2Kazakh Research Institute for Plant Protection and Quarantine
Almaty, Kazakhstan, Kazybek-bi, 1

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Abstract

The movement of viruses through plants plays a key role in viral infection spread and establishment of the virus. Viruses have developed many strategies to ensure their efficient spread through plants and within cells to promote protein expression, replication, and production of the next generation. The movement of viruses is mainly mediated by movement proteins (MP) and to a lesser extent by capsid proteins (CP). Short-distance movement is promoted by the interactions of MP and/or CP with the host cellular cytoskeleton, which directs targeting of viral particles to the plasmodesmata. Plasmodesmata allow free intercellular passage of small molecules and limit movement of viruses due to their exclusion size. Viruses use different strategies to expand the diameter of the plasmodesmataand to enable spread of infectious viral particles over short distances. Viruses have also adapted to interact with particular host factors to promote their long-distance movements through carbohydrate fluxes. Different viral species interact with various host factors to promote their movement. The main plant regulators of viral movement along plants are callose deposits and the RNA interference mechanism. The study of viral movement is important for agriculture, for medicine, and for vector engineering to obtain recombinant products.

Keywords: cell-to-cell movement, movement, movement protein, phloem channels, plant viruses, plasmodesmata, callose

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Author

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DOI: 10.11134/btp.2.2017.4
Khapilina O.N.1, Daniyarov A.Z.1, АmenovА.А.1, Novakovskaya А.P.1, ТurzhanovaА.S.1, Тagimanova D.S.1, Filippova N.I.2, Каlendar R.N.1
National Center for Biotechnology
1National Center for Biotechnology
13/5, Korgalzhyn hwy, Astana,  Kazakhstan
2 Scientific-Production Center of Grain Farming named after A.I. Barayev, Akmola reg., Kazakhstan

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Abstract

Retrotransposon-based molecular markers were used to assess variation in the perennial legumes alfalfa, sweet clover, and lupin from local (Kazakhstan) and world gene pools. Specific retrotransposons were found for species in the Fabaceae family. Conserved regions of LTR retrotransposon sequences were used to design PCR primers to detect polymorphisms by the IRAP method. Universal primers for retrotransposon primer binding site (PBS) sequences were also used. A preliminary screen was used to select the most informative primers that identified up to 80% of polymorphisms. Cluster analysis was carried out to quantify polymorphism and divergence. LTR primers can be used for the simultaneous detection of polymorphic loci that are distributed evenly across the genome. In addition, the study of genetic polymorphism using retrotransposons is distinguished with availability and informative of the method. LTR retrotransposons were employed to study genetic variability and were able to separate the perennial legume varieties according to their relationships and genetic diversity. The results obtained using this method provides a basis for better control of germplasm, future systematic studies, and genetic improvement. This method can also be applied to thestudy of the role of retrotransposons in the genetic variability of species and the dynamics of their genomes.

Keywords: perennial legumes, molecular markers, iPBS, retrotransposons, genotyping.
Abbreviations: LTR, long terminal repeat; PBS, primer binding site; IRAP, inter-retrotransposon amplified polymorphism; REMAP, Retrotransposon microsatellite amplified polymorphism; iPBS, inter-primer binding site polymorphism.

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Author

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DOI: 10.11134/btp.2.2017.5
Chebonenko O.V., AmirkulovaA.Zh., Tursunova A.K., Sapko O.A., Abayldaev A.O., UtarbayevaA.Sh.
M.A. Aitkhozhin institute of molecular biology and biochemistry,
86, Dosmukhamedov str., Almaty, Kazakhstan

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Abstract

Ascorbic acid (AC) is a low-molecular antioxidant that plays a key role in regulating the level of reactive oxygen species (ROS) and products of lipid peroxidation (LPO) in plant cells and also participates in some physiological processes in plants. Ascorbic acid functions to remove many free radicals and to minimize the damaging effects of oxidative stress. Plants resistant to drought and salinity are characterized by higher levels of ascorbic acid, which are actively involved in the regulation of ROS under stress. We investigated the effect of exogenous ascorbic acid at a concentration of 10 mM on the activity of key antioxidant enzymes (AOEs): superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), and pro-oxidant levels (H2O2, LPO) in the potato varieties ‘Axor’ and ‘Orbit’under conditions of salt stress. There were no significant varietal differences in the levels of pro- and antioxidant responses to ascorbic acid treatment. It was shown that ascorbic acid in shoots induced generation of H2O2 and reduced the level of LPO two-fold under salt stress. Ascorbic acid differentially regulates the activities of enzymes under saline conditions. The greatest impact of ascorbic acid was observed on SOD and APX in shoots, with a two-fold increase in their activity. The activity of CAT and POD increased 1.5–2-foldunder the combined effects of ascorbic acid and salinity. Thus, ascorbic acid can regulate the level of response of the antioxidant system of potato plants to salinity. Antioxidant enzymes have an active role in neutralizing ROS and adapting plants to stressful conditions.

Keywords: potato, salinity resistance, ascorbic acid (AC), antioxidant enzymes (AOE), hydrogen peroxide (H2O2), lipid peroxidation (LPO).

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Author

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DOI: 10.11134/btp.2.2017.6
TagimanovaD.1, KhapilinaO.1, AmenovA.1, DanilovaA.2,  Kalendar R.1
1National Center for Biotechnology 
Kоrgalzhyn hwy, 13/5Astana, Kazakhstan
2Altai Botanical Garden, Ridder, Kazakhstan
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Abstract

Roseroot (Rhodiola rosea L.) is a widely used medicinal herb in Russia, Scandinavia, and China. We used intron polymorphism markers for several genes families with high levels of polymorphism to investigate 32 accessions. In addition, we collected plants from 2 separate natural populations in the Altai region (West Altai and South Altai) of Kazakhstan. Universal PCR primer pairs were based on sequenced genes of related Rhodiolaspecies. The intron polymorphism markers were used to assess genetic diversityusing the GenAlex 6.5 program. The discriminatory potential of the selected markers was sufficientto determine the intrapopulation variability of Rhodiola. Observed heterozygosity at the loci averaged 0.235, compared to the expected 0.249. A dendrogram based on genetic distances was calculated from the results and confirmed that the two populations were genetically diverse. An analysis of molecular variance indicated that species level genetic diversity was relatively high (p = 70%) and an analysis using Shannon’s index showed that within and between genetic diversity in roseroot wasapproximately equal. Nei’s genetic distance and unweighted pair-group method with arithmetic averages cluster analysis showed that the two populations formed three major clusters. Understanding the genetic structure of R.rosea L. will improve the conservation and management of this endangered species.

Keywords: Genetic diversity, intron-length polymorphism, Rhodiola rosea, roseroot

 

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Author

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DOI: 10.11134/btp.2.2017.7
Romadanova N.V., Machmutova I.A., Karasholakova L.N., Christenko A.A., Kushnarenko S.V.
National Center for Biotechnology
Institute of Plant Biology and Biotechnology
Almaty, 050040, Kazakhstan

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Abstract

The creation of an in vitro collection of aseptic plants is the first stage for preservation of genetic diversity of barberry in a cryobank and production of mother plantations in natural habitats.Seedlings and plantlets of barberry were obtained from seeds and cuttings, respectively,using biotechnological methodsto produce aseptic plants. Scarification and stratification is required for seeds of some species that are difficult to germinate in wet perlite. Treatment of explants with 0.1% HgCl2 for 8 min is optimal for in vitro culture and produces 46.4%regeneration.In vitro plants were tested for the presence of infection in a specialized 523 medium to detectbacteria and fungi. On average,44.5% of the in vitroshoots in all the tested accessions were affected by endophytic infection. The composition of the nutrient medium was optimized as Murashige-Skoog medium with 166 mg/l CaCl2, 3.7 g/l MgSO4·7H2O, 30 g/l sucrose, 0.8 mg/l 6-benzylaminopurine, 0.02 mg/l indolebutyricacid, 0.1 mg/l gibberellic acid, 2 mg/l calcium pantothenate, 1 mg/l ascorbic acid, 1.75 g/l gelrite, 4 g/l agar, pH 5.7. Amaximum multiplication factorof 4.3 and high quality of regenerated plants were obtained using the optimized medium. A biotechnological regulation was developed to obtain an in vitrocollection of barberry plants anda collection consisting of 51 accessions was created. This collection will serve as the basis for the creation of cryobank of Kazakhstan barberry germplasm and for international exchange of plant resources.

Key words: barberry, seeds, shoots, micropropagation, in vitro collection

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Author

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DOI: 10.11134/btp.2.2017.8
Zhagipar F.S., Nechay N.L., Moldagulova N.B., Yagofarova A.Ya., Berdimuratova K.T., Kakimzhanova A.A.
National center for biotechnology,
Korgalzhin hwy, 13/5, Astana, 010000, Kazakhstan
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Abstract

Oil and oil products are among the main and widespread sources of pollution of water and terrestrial ecosystems. Accidents in oil production can pollute both water and soil ecosystem; in the latter, the pollution can change the soil structure and its redox potential, degrade living conditions of plants and animals, under mine trophic transfer, and block the natural oxygen cycle. A large number of oil fields are situated in the Kazakhstan Republic, especially in the western areas of the country. Oil production and transportation accidents occur regularly and have a severe and deleterious effect on the environment and on wildlife. Therefore, searching for effective bioremediation methods for water and soil is vital. Hydrocarbons can be degraded by microbial activity, for example, by fungal species that can use oil and oil products as a source of carbon and energy. The aim of our work is to characterize the ability of the microscopic fung us Aspergillus terreus 49/10-1 to degrade oil and oil products in soil and water. This strain was isolated from soil of the Caspian Sea and shows high efficiency in bioremediation of oil-contaminated water and soil. More than 89.5% of oil and oil products in water was degraded; in soil, more than 40% was degraded.

Keywords: microscopic fungi, IR spectrometry, degradation, oil

 

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Author

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DOI: 10.11134/btp.2.2017.9
Turgimbayeva A.M.1,2, Abeldenov S.K.1, Akhmetova D.G.3, Saparbayev M.K.4, Ramankulov Y.M.1, Khassenov B.B.1
1National Center for Biotechnology
Korgalzhyn hwy, 13/5, Astana, 010000, Kazakhstan
2L.N. Gumilyov Eurasian National University
Satpayev Street, 2, Astana, 010000, Kazakhstan
3Republican Diagnostic Center
Syganak Street, 2, Astana, 010000, Kazakhstan
4Institute of Gustav Roussy,
CNRS UMR 8200, 114 Rue Edouard Vaillant, Villejuif, 94805, France

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Abstract

Studies on bacterial DNA repair mechanisms have historically been carried out in Escherichia coli as the model system. However, evidence is accumulating that DNA repair mechanisms in other bacterial species may differ fundamentally from those of E. coli. Pathogens such as Mycobacterium tuberculosis, Helicobacter pylori, and Staphylococcus aureus have evolved various DNA repair mechanisms that help them to persist. When bacterial pathogens enter the human body they are exposed to a range of host defense mechanisms, such as the formation of reactive oxygen species and reactive nitrogen intermediates that can induce mutations in their genomes. Bacterial infections can induce a range of pathogenic diseases, and each of the causative bacterial species has characteristic DNA repair mechanisms. The study of the functions and biological roles of DNA repair enzymes is very important for understanding bacterial persistence in the human body. Moreover, repair enzymes might be potentially new targets for therapeutic agents. In this study, the DNA repair mechanisms of various human pathogens are described.

Keywords: DNA repair, Mycobacterium tuberculosis, Helicobacter pylori, Staphylococcus aureus.

 

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Author

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DOI: 10.11134/btp.2.2017.10
Akishev Zh.D.1,2, Abdullayeva A.N.1,2, AbeldenovS.K.1, KhassenovB.B.1
1National Center for Biotechnology,
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
2L.N.Gumilyov Eurasian National University,
2, Kanysh Satpayev str., Astana, 01000
8, Kazakhstan
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Abstract

Bovine chymosin (EC 3.4.23.4), which belongs to a family of aspartic proteases, is mainly derived from mammalian gastric mucosal cells of the abomasum of unweaned calves. Chymosin contains two residues of Asp in an active site, which catalyze the selective cleavage of a peptide bond in κ-casein between Phe105 and Met106. This catalytic process yields insoluble para-κ-casein, which causes milk coagulation. In this study, we sought to identify an alternative milk coagulant that is safe and efficient and, at the same time, can produce cheese with a good taste. Bovine prochymosin B was chosen and constitutively expressed at a high level in Pichia pastoris. The recombinant chymosin protein was expressed as a secretory form and was found to exhibit milk-clotting activity. It was stable at 25–50°C and had optimal activity at 37°C and pH 6.27. The activity of the recombinant chymosin was activated by cations such as Ca2+, Mg2+, Fe3+, Cd2+, Co2+, and Mn2+, but inhibited by Ni2+, and was not affected by Na+, K+, Cs2+, and Li+. These results suggest that recombinant bovine chymosin is an acid milk coagulant, and that it could be a safe and efficient enzyme suitable for use in cheese production.

Keywords: bovine, chymosin, casein, milk coagulation, Pichia pastoris.

 

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Author

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DOI: 10.11134/btp.2.2017.11
Baltin K.K., AkishevZh.D., Abeldenov S.K., Silayev D.V., Khassenov B.B.
National Center for Biotechnology,
13/5, Korgalzhyn hwy, Astana, Kazakhstan

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Abstract

Recombinant β-galactosidase from Streptococcus thermophilus was successfully expressed in Escherichia coli, purified, and biochemically characterized. The gene encoding β-galactosidase was amplified from the genomic DNA of St. thermophilus and cloned into the expression vector pET-28c (+). Using the recombinant vector, a BL21 (DE3)/pLacZST strain-producer was obtained with overexpression of the gene. Optimal culture parameters for producing recombinant β-galactosidase were determined. The recombinant β-galactosidase had an activity of 19 units/mg. Biochemical characterization of recombinant β-galactosidase showed that the enzyme had maximum activity at pH 9.0 and temperature of 60°C. Analysis of the kinetics of lactose hydrolysis gave a Michaelis constant Kmof 10.12 ± 2.5 mM and a limiting value of the initial rate of the enzymatic reaction Vmaxof 0.47 ± 0.027 mM/min. The β-galactosidase has been used in experiments that simulate commercial production conditions, to producea glucose-galactose syrup.

Keywords: β-galactosidase, Streptococcus thermophilus, genomic DNA, lactose.

 

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Author

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DOI: 10.11134/btp.2.2017.12
Li P., Abeldenov S., Khassenov B.
National Center for Biotechnology
13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan

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Abstract

Isolation of thermostable DNA polymerase from Thermusaquaticus was a significant stage in molecular biology and laboratory performance. After Taq DNA polymerase was discovered PCR method became widespread among laboratories around the world. Taq DNA polymerase was the first tool for fast and highly specific amplification of targeted nucleotide sequences. Thus, thermostable polymerases became vital for laboratory performance.

We managed to express and purify recombinant Tth DNA polymerase that has both polymerase and reverse transcriptase activities. At first we cultivated Thermusthermophilus strain HB8 and isolated genomic DNA. The gene was amplified and cloned into expression plasmid vector pET-28c(+) under T7 promoter. E.coli cells BL-21(DE3) were transformed with obtained recombinant vector and cultivated with kanamycin antibiotic in LB-broth. Induction was started with IPTG. Cells were disrupted by lysozyme and ultrasonication. Liquid fraction was loaded into sepharose column.

Obtained purified enzyme is highly thermostable which was tested in a condition of high temperature and are able to preserve polymerase activity even after heating at 95℃ during 30 minutes. Recombinant Tth polymerase has 95% SDS-PAGE purity. Also we have managed to made different storage and reaction buffers (with various concentrations of salts, stabilizers and detergents) in order to determine the best combination.

Keywords: Tth, Thermusthermophilus, recombinant protein, E.coli, polymerase, enzyme.

 

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