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Background Since the beginning of the use of reporter proteins for expression analysis, a variety of approaches have been developed and proposed; both qualitative and quantitative. The lack of simple methods for direct observation of gene expression in living organisms makes it necessary to continue to propose new methods. In this work, we consider a method for the quantitative analysis of the expression of thermostable lichenase from Clostridium thermocellum used as a sensitive reporter protein. Results In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusions The specific interaction between the dye Congo red and $$\beta$$ β -d-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

In order to obtain high yields of winter durum wheat, it is necessary not only to use advanced agricultural technologies, but also to sow varieties resistant to major diseases. One of these diseases that cause great harm to grain crops is Septoria leaf blotch. The current paper has presented the study results of collection material of Russian and foreign winter durum wheat according to the efficiency of the Septoria leaf blotch resistance gene Stb2 in the North Caucasus region. The purpose of the current study was to identify the Septoria leaf blotch resistance gene Stb2 in the collection material of winter durum wheat. The study was carried out in 2017–2018 in the laboratory of marker breeding of the FSBSI “ARC “Donskoy” in Zernograd. In the course of the study there have been applied such methods as the isolation of genomic DNA by the CTAB-method, PCR-analysis with the molecular SSR marker WMS533, electrophoresis on 2% agarose gel, analysis of electrophoresis gels in the Bio-Rad Image Lab 6.0 software, general data analysis by Microsoft Excel. The study has shown that 29 samples out of 185 analyzed ones had the Septoria leaf blotch resistance gene Stb2, they are the lines L3557 h42, L3182 h7, L3335 h29, L3336 h43, L3408 h218 (Russia, FSBSI “Research Center of Plants named after P. P. Lukyanenko”), K-61620 (Russia), K-54455, DF 917 (Romania), K-61924, K-59179, K-51863, K-61869 (Moldova), K-63871 (Turkey), Kharkovskaya 909 (Ukraine), MVTD-15-99 (Hungary), 2196-1/04, 922/93, 589/13, 655/13, 683/13, 145/14, 322/14, 484/14, Gordeiform 1424/82 (Russia, FSBSI “ARC “Donskoy”), Beliy Parus (Ukraine), Belgorodskaya Yantarnaya (LLC “Sativa” in Belgorod, Russia), IR named after V. Ya. Yuriev (Kharkov, Ukraine)), Zhivitsa (Russia, Prikumskaya OSS), Pandur (Romania) and Elidur (Romania). There have been recommended using the identified samples of winter durum wheat with the Septoria leaf blotch resistance gene Stb2 in breeding programs.

The purpose of the current work was to study the winter wheat varieties artificially infected with the North Caucasian population of brown rust to identify a damage degree. The study was carried out in two stages on two sets (8 and 10) of varieties with different level of resistance to the pathogen. The trials have established a high damage degree of rust on susceptible varieties, which resulted in productivity decrease from 16.3 to 32.2%. Among the varieties with the maximum damage degree, there was identified the most resistant variety ‘Tarasovskaya 29’ which, when completely (100%) damaged by the disease, showed a minimum productivity decrease, no more than 10.6%. The varieties being poorly susceptible to brown leaf rust reduced the productivity from 11.2% to 20.7%. The smaller indices of productivity decrease among this group were shown by the variety ‘Spartak’. Among the five varieties that showed moderate leaf rust damage, there were different degrees of productivity decrease. The variety ‘Donskaya Yubileynaya’ artificially infected with brown rust, showed a minimal productivity decrease and its structural elements, and it could be identified as the leaf rust resistant variety like the variety ‘Tarasovskaya 29’. When predicting the brown leaf rust epiphytoty, the susceptible and half-resistant wheat varieties require obligatory protection with fungicides.

Fatty acid desaturases perform a key role in the maintenance of cell membrane homeostasis. They convert fatty acids of membrane lipids from saturated to unsaturated. The modulation of fatty acid unsaturation in cell membranes is one of the mechanisms of plant adaptation to traumatic environmental factors, both biotic and abiotic. The study of the mechanisms of the functioning of desaturases, as well as their localization and characteristics of their gene expression, carry out a fundamental role in our understanding of the processes of plant adaptation. In addition to their fundamental importance, such studies have an applied character. They will expand the potential of desaturases and put in place the foundations of a modern generation of transgenic plants, including those derived from genome-editing technologies. As a result, these studies deliver a broad benefit to humanity in the creation of stress-resistant crops or the biofortification of agricultural crops.

Transient heterologous gene expression in two model plant species, Nicotiana benthamiana and N. excelsior, has been used to study the localization of the heterologous Δ9 acyl-lipid desaturase (Δ9 desaturase) of Synechococcus vulcanus in different cell compartments and its functional activity in the cases of the cytosol, chloroplast, and endoplasmic reticulum (ER) localization. The functional activity and substrate specificity of the heterologous desaturase under the conditions of transient expression have been confirmed by comparison of fatty acid (FA) profiles. The Δ9 desaturase, responsible for the synthesis of oleic and palmitoleic acids, has also been shown to strongly promote the accumulation of polyunsaturated FAs. The results convincingly demonstrate that the Δ9 desaturase of the thermophilic cyanobacterium transiently expressed in two Nicotiana species considerably alters lipid metabolism in their leaves towards a higher FA unsaturation. The functional activity of Δ9 desaturase depends on both the model plant species, N. benthamiana or N. excelsior, and the cellular localization of the enzyme. The method of transient expression of heterologous genes in plants is highly effective, inexpensive, and not time-consuming, which makes it attractive for estimating the functional activity and/or substrate specificity of heterologous desaturases.

BackgroundMolecular biology has always shown some similarities with computer science. So, considering transient expression, one can see an analogy with a DDoS attack on a computing system. Like the DDoS attack, transient expression can carry a payload. In particular, analysis of the structure of cell mechanisms and signal amplification in the study of very subtle mechanisms of regulation.ResultsA new vector system for transient expression in plants is described; this system is intended for quantitative analysis of the contribution of regulatory elements to transcription and translation efficiencies. The proposed vector comprises two expression cassettes carrying reporter genes (of the Clostridium thermocellum thermostable lichenase and E. coli

The paradox of misfit between the levels of mRNAs and their protein products in the eukaryotic cells, including plant cells, encountered by researchers, direct their efforts towards the study into fine mechanisms of translation. Translation is an intricate biological process with numerous players, including mRNAs, tRNAs, ribosomes, and manifold protein factors. Certainly, each of them is important for efficient translation. However, the mRNAs itself contain numerous regulatory elements, such as 5'UTR, the context around the AUG start codon, and codon composition; each element separately or in combination can determine the fate of an individual mRNA in translational process. The previous reviews mainly focus on individual key stages in translation or the aspects of its control. Our goal here is (i) to summarize the recent data on the specific structure–function features of plant mRNAs and their correlation with translational efficiency; (ii) to brief new experimental and theoretical approaches to gaining the insight into the complex network of translation; and (iii) to assess the relevance of this knowledge to both the plant functional genomics and biotechnological application.

On the fields with infectious backgrounds more than one thousands of varieties and samples of collection and breeding material of winter wheat are annually estimated for resistance to six diseases (brown and yellow rust, leaf blotch, powdery mildew, loose and kernel smut). Among the collection material there have been identified the samples tolerant and resistant to all studied diseases as well as the samples with complex resistance to 3–6 pathogens (e. g. “Maris Kinsman”, “Tucan”, “Liwilla”, “KS93V76”, “PL 178383” and others). Such varieties of the Inter-Station Variety Testing (ISVT) from Russia, Ukraine, Germany, France as “Etana”, “Kurs”, “Rotaks”, “Solokha”, “Shestopalovka” cannot be artificially infected with the diseases. There has been identified a small percentage among the present varieties and collection samples tolerant to such diseases as powdery mildew, leaf blotch and kernel smut. The winter soft wheat varieties developed in the FSBSI “ARC “Donskoy” both widely cultivated in production, and introduced for study in the SVT in recent years, are characterized by a high resistance to 2–4 studied diseases. These are the varieties “Tanais”, “Rostovchanka 7”, “Aksiniya”, “Nakhodka” which are recommended to plant after fallow forecrops and the varieties “Krasa Dona”, “Ermak”, “Donskoy Syurpriz”, “Don 107”, “Izyuminka”, “Asket”, “Lidiya” which are recommended to plant after non-fallow forecrops. The identified resistant varieties are recommended for wide cultivation, and the sources of resistance to a complex of the main harmful diseases on the Nizhny Don can be used in the breeding programs on immunity.

The transcript Euonymus europaeus was collected de novo. A comparative analysis of transcriptomes from the stage of the globular embryo and the mature fruit made it possible to identify key DGAT genes at the contrasting stages of the development of E. europaeus fruits.

Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase, EC 3.2.1.73 (P29716)) has been tested for the insertion of two model fluorescent proteins (EGFP and TagRFP) into two regions of this enzyme. Functional folding of the resulting proteins was confirmed by retention of lichenase activity and EGFP and TagRFP fluorescence. These results convincingly demonstrate that (i) the two experimentally selected lichenase loop regions may serve as the areas for domain insertion without disturbing enzyme folding in vivo; (ii) lichenase permits not only single but also tandem insertions of large protein domains. High specific activity, outstanding thermostability, and efficient in vitro refolding of thermostable lichenase make it an attractive new host protein for the insertional fusion of domains in the engineering of multifunctional proteins.

The contribution of nucleotide composition of mRNA 5'-region to the efficiency of expression at transcriptional and translational levels was studied in transgenic tobacco plants (Nicotiana tabacum L., cultivar Petit Havana) using a thermostable lichenase reporter gene. Synthetic sequence that contains CG-rich motifs, typical for 5'-region of plant genes, identified in silico, was constructed. Transgenic plant lines of N. tabacum were obtained; they contain thermostable lichenase reporter gene that is under control of the constitutive 35S RNA CaMV promoter and additional regulatory element: synthetic CG-rich sequence, which functions as the 5'-UTR (untranslated region) of the reporter gene mRNA or as 5'-region of the hybrid gene coding sequence, wherein the synthetic sequence is fused with the sequence of the reporter gene in its reading frame. Results of the comparative analysis of mRNA and protein levels in the obtained lines of transgenic plants showed that the synthetic CG-rich sequence significantly increases the level of transcription of the reporter gene and appear to have no negative effect on the efficiency of reporter mRNA translation, which may be due to the peculiarities of its nucleotide composition and structure, namely, due to the presence of motifs that are specific for 5'-regions of plant genes, as well as due to the properties of the secondary structure— the absence of hairpin structures with a high energy of formation. It was experimentally confirmed for the first time that the 5'-region of the genes with a high content of CpG dinucleotides can help to increase the transcription level of genes in plants.

The control of translation in the course of gene expression regulation plays a crucial role in plants’ cellular events and, particularly, in responses to environmental factors. The paradox of the great variance between levels of mRNAs and their protein products in eukaryotic cells, including plants, requires thorough investigation of the regulatory mechanisms of translation. A wide and amazingly complex network of mechanisms decoding the plant genome into proteome challenges researchers to design new methods for genome-wide analysis of translational control, develop computational algorithms detecting regulatory mRNA contexts, and to establish rules underlying differential translation. The aims of this review are to (i) describe the experimental approaches for investigation of differential translation in plants on a genome-wide scale; (ii) summarize the current data on computational algorithms for detection of specific structure–function features and key determinants in plant mRNAs and their correlation with translation efficiency; (iii) highlight the methods for experimental verification of existed and theoretically predicted features within plant mRNAs important for their differential translation; and finally (iv) to discuss the perspectives of discovering the specific structural features of plant mRNA that mediate differential translation control by the combination of computational and experimental approaches.

A recombinant DNA in which the interferon αA (IFN-αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN-αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN-αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.