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Ignatov K.B.,Russian Academy of Sciences | Barsova E.V.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Fradkov A.F.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Blagodatskikh K.A.,All Russia Institute of Agricultural Biotechnology | And 2 more authors.
BioTechniques | Year: 2014

The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Taq DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.


Vysotskii D.A.,All Russia Institute of Agricultural Biotechnology | Kostina M.B.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Roslyakova T.,All Russia Institute of Agricultural Biotechnology | Leonova T.,All Russia Institute of Agricultural Biotechnology | And 3 more authors.
Russian Journal of Plant Physiology | Year: 2012

Members of the 14-3-3 protein family are known to be important regulators of plant primary metabolism, hormonal signal transduction, and ion homeostasis. We identified nine isoforms of 14-3-3 genes of Thellungiella salsuginea, an extremophile relative of Arabidopsis thaliana. All the identified isoforms were designated according to their Arabidopsis orthologs: Chi, Omega, Psi, Phi, Upsilon, Lambda, Mu, Epsilon, and Omicron. Comparison of the deduced amino acid sequences reveals high degree of identity between the members of this protein family. Isoforms, designated as Ts14-3-3 Chi, Omicron, and Mu, display noticeable differences in their C-terminal domain as compared to their Arabidopsis homologs. Phylogenetic analysis demonstrated that the identified isoforms split into two groups, epsilon and non-epsilon, according to the common classification of the 14-3-3 family genes. The Thellungiella 14-3-3 isoforms are differentially expressed in various plant tissues, and real-time RT-PCR revealed that most of the isoforms are highly expressed even under normal growth conditions. In response to abiotic stress, low temperatures and high concentrations of salts, 14-3-3 genes exhibited different expression patterns. Our data suggest that, due to the high expression levels of the 14-3-3 genes, Thellungiella plants are likely pre-adapted to the stress conditions. Differences between the C-terminal domains of some Thellungiella 14-3-3 proteins and their Arabidopsis homologs may result in differences in target protein specificity. © 2012 Pleiades Publishing, Ltd.


Mitiouchkina T.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Skachkova T.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Shulga O.,All Russia Institute of Agricultural Biotechnology | Dolgov S.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry
Acta Horticulturae | Year: 2011

The most promising strategy to create virus resistant plants today is gene silencing technology called RNA interference. The essence of RNA-induced gene silencing is the delivery of double-stranded RNA into an organism or cell, inducing a sequence-specific RNA degradation mechanism that effectively silences a targeted gene. For this purpose we transferred the Chrysanthemum virus B coat protein gene sequences into specific vector pKannibal in order to generate small double stranded RNA complementary to this sequence and obtained four transgenic lines of chrysanthemum 'White Snowdon'.


PubMed | Russian Academy of Sciences, All Russia Institute of Agricultural Biotechnology, University of Stockholm and RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry
Type: Journal Article | Journal: BioTechniques | Year: 2014

The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Taq DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.


Vysotskii D.A.,All Russia Institute of Agricultural Biotechnology | de Vries-van Leeuwen I.J.,CSIC - Biological Research Center | Souer E.,CSIC - Biological Research Center | Babakov A.V.,All Russia Institute of Agricultural Biotechnology | de Boer A.H.,CSIC - Biological Research Center
Plant signaling & behavior | Year: 2013

ABF transcription factors are the key regulators of ABA signaling. Using RACE-PCR, we identified and sequenced the coding regions of four genes that encode ABF transcription factors in the extremophile plant Thellungiella salsuginea, a close relative of Arabidopsis thaliana that possesses high tolerance to abiotic stresses. An analysis of the deduced amino acid sequences revealed that the similarity between Thellungiella and Arabidopsis ABFs ranged from 71% to 88%. Similar to their Arabidopsis counterparts, Thellungiella ABFs share a bZIP domain and four conservative domains, including a highly conservative motif at the C-terminal tail, which was reported to be a canonical site for binding by 14-3-3 regulatory proteins. Gene expression analysis by real-time PCR revealed a rapid transcript induction of three of the ABF genes in response to salt stress. To check whether Thellungiella ABF transcription factors can interact with abundant 14-3-3 proteins, multiple constructs were designed, and yeast two-hybrid experiments were conducted. Six of the eight tested Ts14-3-3 proteins were able to bind the TsABFs in an isoform-specific manner. A serine-to-alanine substitution in the putative 14-3-3 binding motif resulted in the complete loss of interaction between the 14-3-3 proteins and the ABFs. The role of 14-3-3 interaction with ABFs in the salt and ABA signaling pathways is discussed in the context of Thellungiella survivability.


Krivosheeva A.B.,RAS Timiryazev Institute of Plant Physiology | Varlamova T.V.,All Russia Institute of Agricultural Biotechnology | Yurieva N.O.,Moscow Institute of Physics and Technology | Sobol'kova G.I.,RAS Timiryazev Institute of Plant Physiology | And 2 more authors.
Russian Journal of Plant Physiology | Year: 2014

Transformation by genes of various ion transporters is a tool to study plant salt tolerance mechanisms. In this work, the gene of a vacuolar NHX antiporter HvNHX3 was introduced in two potato Solanum tuberosum L. cultivars, Yubiley Zhukova and Skoroplodny-7. The binary vector pCambia-HvNHX3 was made and used for transformation. This vector carries HvNHX3 and NPTII for kanamycin selection each driven by 35S promoter from cauliflower mosaic virus. The presence of the target gene HvNHX3 and its mRNA were confirmed in 35 out of 48 kanamycin-resistant transformants. The growth parameters of 13 transgenic lines were measured in control and NaCl-containing media. The transformation of a relatively salt-tolerant cv. Yubiley Zhukova did not significantly change its growth characteristics and salt tolerance. In control conditions, most transgenic lines of cv. Skoroplodny-7 had the larger biomass and height. Unlike untransformed plants, they rooted and grew on 100 mM NaCl, indicating their greater salt tolerance. © Pleiades Publishing, Ltd., 2014.


Vysotskii D.A.,All Russia Institute of Agricultural Biotechnology | de Vries-van Leeuwen I.J.,VU University Amsterdam | Souer E.,VU University Amsterdam | Babakov A.V.,All Russia Institute of Agricultural Biotechnology | de Boer A.H.,VU University Amsterdam
Plant Signaling and Behavior | Year: 2013

ABF transcription factors are the key regulators of ABA signaling. Using RACE-PCR, we identified and sequenced the coding regions of four genes that encode ABF transcription factors in the extremophile plant Thellungiella salsuginea, a close relative of Arabidopsis thaliana that possesses high tolerance to abiotic stresses. An analysis of the deduced amino acid sequences revealed that the similarity between Thellungiella and Arabidopsis ABFs ranged from 71% to 88%. Similar to their Arabidopsis counterparts, Thellungiella ABFs share a bZIP domain and four conservative domains, including a highly conservative motif at the C-terminal tail, which was reported to be a canonical site for binding by 14-3-3 regulatory proteins. Gene expression analysis by real-time PCR revealed a rapid transcript induction of three of the ABF genes in response to salt stress. To check whether Thellungiella ABF transcription factors can interact with abundant 14-3-3 proteins, multiple constructs were designed, and yeast two-hybrid experiments were conducted. Six of the eight tested Ts14-3-3 proteins were able to bind the TsABFs in an isoform-specific manner. A serine-to-alanine substitution in the putative 14-3-3 binding motif resulted in the complete loss of interaction between the 14-3-3 proteins and the ABFs. The role of 14-3-3 interaction with ABFs in the salt and ABA signaling pathways is discussed in the context of Thellungiella survivability. © 2013 Landes Bioscience.

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