Laboratory of Plant Biotechnology

NamePositionContacts
Yaroslav Buryanov, D.Sc, professordepart. dir.buryanov@bibch.ru+7(4967)73-09-21#3228
Natalia Zakharchenko, Ph.D.s. r. f.zachar@bibch.ru
Elena Rukavtsova, D.Scs. r. f.ruk@bibch.ru+7(27)330970
Anna Lebedeva, Ph.D.s. r. f.aanlebedeva@mail.ru
Taras Shevchuk, D.Scs. r. f.shevchuk@bibch.ru
Olga Dyachenko, Ph.D.r. f.ovdyachenko@bibch.ru
Valeriya Alekseeva, Ph.D.r. f.lera@bibch.ru
Svetlana Pigoleva, Ph.D.r. f.svoil@rambler.ru

Former members:

Sergey Tarlachkoveng.sergey@tarlachkov.ru

Selected publications

  1. Kolachevskaya O.O., Alekseeva V.V., Sergeeva L.I., Rukavtsova E.B., Getman I.A., Vreugdenhil D., Buryanov Y.I., Romanov G.A. (2015). Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro. J Integr Plant Biol 57 (9), 734–44 [+]

    Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuberization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhancement of auxin content.

    ID:1437
  2. Бурьянов Я.И. (2015). Адаптивная эпибиохимия и эпигенетика. Биохимия 80 (9), 1376–1390 [+]

    Enzymatic reactions of post-synthetic modification of macromolecules occur in the cells of all organisms. These reactions, which can be designated as epibiochemical, are of a special type and, as discriminated from reactions with low molecular weight substrates, occur on the level of biopolymers, causing their covalent modification. The majority of epibiochemical modifications of proteins, DNA, and RNA are reversible and are carried out by modification transferases and de-modification enzymes, respectively. Epibiochemical, i.e. those located above the low molecular weight metabolites, modifications of proteins and nucleic acids perform various functions, including participation in molecular mechanisms of adaptive epigenetic heredity. This paper presents an overview of some adaptive epibiochemical modifications of macromolecules and the adaptive epigenetic processes on their basis. The features of epigenetic inheritance of acquired characteristics and the limits of biological evolution are discussed.

    ID:1439
  3. Rukavtsova E.B., Rudenko N.V., Puchko E.N., Zakharchenko N.S., Buryanov Y.I. (2015). Study of the immunogenicity of hepatitis B surface antigen synthesized in transgenic potato plants with increased biosafety. J. Biotechnol. 203, 84–8 [+]

    Oral immunogenicity of the hepatitis B surface antigen (HBsAg) synthesized in the tubers of marker-free potato plantshas been demonstrated. Experiments were performed in the two groups of outbred NMRI mice. At the beginning of investigations, the mice of experimental group were fed the tubers of transgenic potato synthesizing the HBsAg three times. The mice of control group were fed nontransgenic potato. Intraperitoneal injection of the commercial vaccine against hepatitis B (0.5μg/mouse) was made on day 71 of the experiment. Enzyme-linked immunoassay (ELISA) of the serum of immunized animals showed an increase in the level of HBsAg antibodies significantly above the protective value, which was maintained for 1 year after the immunization. In 1 year, the experimental group of mice underwent additional oral immunization with HBsAg-containing potato tubers. As a result, the level of antibodies against the HBsAg increased and remained at a high protective level for several months. The findings show the possibility of usingtransgenic plants as a substance for obtaining a safe edible vaccine against hepatitis B.

     

    ID:1436
  4. Захарченко Н.С., Лебедева А.А., Фурс О.В., Рукавцова Е.Б., Шевчук Т.В., Дьяченко О.В., Бурьянов Я.И. (2015). Новая экспрессионная система для повышенного синтеза антимикробного пептида цекропина Р1 в растениях. Физиология растений 62 (4), 571–578 ID:1438
  5. Dyachenko O.V., Tarlachkov S.V., Marinitch D.V., Shevchuk T.V., Buryanov Y.I. (2014). Expression of exogenous DNA methyltransferases: application in molecular and cell biology. Biochemistry Mosc. 79 (2), 77–87 [+]

    DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

    ID:1451
  6. Dyachenko O.V., Schevchuk T.V., Kretzner L., Buryanov Y.I., Smith S.S. (2010). Human non-CG methylation: are human stem cells plant-like? Epigenetics 5 (7), 569–72 [+]

    Non-CG methylation is well characterized in plants, where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in animals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in animals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.

    ID:1452
  7. Rukavtsova E.B., Gaiazova A.R., Chebotareva E.N., Burianova Ia.I. (2009). Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen. Genetika 45 (8), 1055–60 [+]

    The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01-0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.

    ID:1442
  8. Rukavtsova E.B., Chebotareva E.N., Rudenko N.V., Buryanov Y.I. (2009). Immunogenicity of biologically safe potato tubers synthesizing hepatitis B surface antigen. Dokl. Biol. Sci. 437, 110–2 ID:1440
  9. Rukavtsova E.B., Alekseeva V.V., Buryanov Ia.I. (2009). The use of RNA interference for the metabolic engineering of plants. Bioorg. Khim. 36 (2), 159–69 [+]

    The metabolic engineering of plants is aimed at the realization of new biochemical reactions by transgenic cells. These reactions are determined by enzymes encoded by foreign or self-modified genes. Plants are considered to be the most interesting objects for metabolic engineering. Although they are characterized by the same pathways for the synthesis of basic biological compounds, plants differ by the astonishing diversity of their products: sugars, aromatic compounds, fatty acids, steroid compounds, and other biologically active substances. RNA interference aimed at modifying metabolic pathways is a powerful tool that allows for the obtainment of plants with new valuable properties. The present review discusses the main tendencies for research development directed toward the obtainment of transgenic plants with altered metabolism.

    ID:1441
  10. Rukavtsova E.B., Zakharchenko N.S., Pigoleva S.V., Yukhmanova A.A., Chebotareva E.N., Buryanov Y.I. (2009). Obtaining marker-free transgenic plants. Dokl. Biochem. Biophys. 426, 143–6 ID:1443
  11. Alekseeva V.V., Rukavtsova E.B., Golubchikova Iu.S., Burianov Ia.I. (2009). Inhibition of agrobacterial oncogenes expression by means of antisense RNA. Mol. Biol. (Mosk.) 42 (1), 172–7 [+]

    Plant's infection with soil bacteria Agrobacterium tumefaciens lead to tumour formation, so called crown galls. The reason of tumorigenesis is integration of agrobacterial genes for phytohormone synthesis auxins and cytokinins in plant genome, the most important of them are iaaM and ipt. Obtaining of transgenic plants able to inhibit these genes expression, creates conditions for producing of plants resistant to crown gall disease. With this purpose single and double transformants of tobacco plants with antisense copies of iaaM and ipt genes under the control of single and double promoters of 35S RNA of cauliflower mosaic virus (CaMV 35S and CaMV 35SS) were produced. Infection with virulentA. tumefaciens strains C58 (pTiC58) and A6 (pTiA6) of all types transgenic plants with antisense oncogenes copies showed essential but incomplete inhibition of these genes expression. After agrobacterial transformations of transgenic plants only "weakened" tumours of various morphology, able to regenerate the whole plants, were formed. The analysis data of inhibition of iaaM and ipt genes expression in formed tumour cells were presented. The results indicate perspective RNA-interference strategy for producing of plants resistant to agrobacterial crown gall disease.

    ID:1444
  12. Zakharchenko N.S., Rukavtsova E.B., Gudkov A.T., Yukhmanova A.A., Shkolnaya L.A., Kado C.I., Buryanov Y.I. (2009). Expression of the artificial gene encoding anti-microbial peptide cecropin P1 increases the resistance of transgenic potato plants to potato blight and white rot. Dokl. Biol. Sci. 415, 267–9 ID:1446
  13. Kalyaeva M.A., Ivanova E.G., Doronina N.V., Zakharchenko N.S., Trotsenko Y., Buryanov Y. (2009). Stimulation of wheat morphogenesis in vitro by methanotrophic bacteria. Dokl. Biol. Sci. 388, 76–8 ID:1462
  14. Semenyuk E.G., Stremovskiy O.A., Edelweiss E.F., Shirshikova O.V., Balandin T.G., Buryanov Y.I., Deyev S.M. (2007). Expression of single-chain antibody-barstar fusion in plants. Biochimie 89 (1), 31–8 [+]

    We successfully cloned and expressed a single-chain antibody (425scFv), that is directed to human epidermal growth factor receptor HER1 (EGFR) in transgenic tobacco plants as a fusion with bacterial barstar gene (425scFv-barstar). Plant-produced recombinant 425scFv-barstar was recovered using barstar-barnase system. Based on barstar-barnase affinity, during purification of the plant-produced 425scFv-barstar, we generated bispecific scFv-antibody heterodimers from individual single-chain fragments initially produced in different host systems with binding activity to both HER1 and HER2/neu tumor antigens. We demonstrated by flow cytometry and indirect immunofluorescent microscopy that both the components of heterodimer retain its specific cell-binding activity.

    ID:1453
  15. Dyachenko O.V., Zakharchenko N.S., Shevchuk T.V., Bohnert H.J., Cushman J.C., Buryanov Y.I. (2006). Effect of hypermethylation of CCWGG sequences in DNA of Mesembryanthemum crystallinum plants on their adaptation to salt stress. Biochemistry Mosc. 71 (4), 461–5 [+]

    Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5'-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.

    ID:1454
  16. Zakharchenko N.S., Rukavtsova E.B., Gudkov A.T., Burianov Ia.I. (2005). Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1. Genetika 41 (11), 1445–52 [+]

    Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F1 and F2 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.

    ID:1447
  17. Buryanov Y.I., Shevchuk T.V. (2005). DNA methyltransferases and structural-functional specificity of eukaryotic DNA modification. Biochemistry Mosc. 70 (7), 730–42 [+]

    Properties of the main families of mammalian, plant, and fungal DNA methyltransferases are considered. Structural-functional specificity of eukaryotic genome sequences methylated by DNA methyltransferases is characterized. The total methylation of cytosine in DNA sequences is described, as well as its relation with RNA interference. Mechanisms of regulation of expression and modulation of DNA methyltransferase activity in the eukaryotic cell are discussed.

    ID:1456
  18. Buryanov Y., Shevchuk T. (2005). The use of prokaryotic DNA methyltransferases as experimental and analytical tools in modern biology. Anal. Biochem. 338 (1), 1–11 [+]

    Prokaryotic DNA methyltransferases (MTases) are used as experimental and research tools in molecular biology and molecular genetics due to their ability to recognize and transfer methyl groups to target bases in specific DNA sequences. As a practical tool, prokaryotic DNA MTases can be used in recombinant DNA technology for in vitro alteration and enhancing of cleavage specificity of restriction endonucleases. The ability of prokaryotic DNA MTases to methylate cytosine residues in specific sequences, which are also methylated in eukaryotic DNA, makes it possible to use them as analytical reagent for determination of the site-specific level of methylation in eukaryotic DNA. In vivo DNA methylation by prokaryotic DNA MTases is used in different techniques for probing chromatin structure and protein-DNA interactions. Additional prospects are opened by development of the methods of DNA methylation targeted to predetermined DNA sequences by fusion of DNA MTases to DNA binding proteins. This review will discuss the application of prokaryotic DNA MTases of Type II in the methods and approaches mentioned above.

    ID:1457
  19. Shevchuk T., Kretzner L., Munson K., Axume J., Clark J., Dyachenko O.V., Caudill M., Buryanov Y., Smith S.S. (2005). Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells. Nucleic Acids Res. 33 (19), 6124–36 [+]

    Several reports suggest that C(m)CWGG methylation tends not to co-exist with (m)CG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII-GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of C(m)CWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied C(m)CWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring C(m)CWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C(m)CWGG in its promoter. Kinetic studies suggested that an adjacent C(m)CWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C(m)CWGG methylation does not exert a significant effect on CG methylation in human kidney cells.

    ID:1455
  20. Clark J., Shevchuk T., Swiderski P.M., Dabur R., Crocitto L.E., Buryanov Y.I., Smith S.S. (2005). Construction of ordered protein arrays. Methods Mol. Biol. 300, 325–48 [+]

    Artificially ordered protein arrays provide a facile approach to a variety of problems in biology and nanoscience. Current demonstration systems use either nucleic acid tethers or methyltransferase fusions in order to target proteins or peptides of interest to nucleic acid scaffolds. These demonstrations point to the large number of useful devices and assemblies that can be envisioned using this approach, including smart biological probes and drug delivery systems. In principle, these systems are now capable of imitating the earliest forms of prebiotic organisms and can be expected to reach the complexity of a small virus in the near future. Third-generation methyltransferase inhibitors provide an example of a smart chemotherapeutics that can be constructed with this approach. We describe the use of mechanistic enzymology, computer-aided design, and microfluidic chip-based capillary electrophoresis in assessing the final assembly and testing of designs of this type.

    ID:1458
  21. Shulga N.Y., Rukavtsova E.B., Krymsky M.A., Borisova V.N., Melnikov V.A., Bykov V.A., Buryanov Y.I. (2004). Expression and characterization of hepatitis B surface antigen in transgenic potato plants. Biochemistry Mosc. 69 (10), 1158–64 [+]

    Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

    ID:1448
  22. Marinitch D.V., Vorobyev I.A., Holmes J.A., Zakharchenko N.S., Dyachenko O.V., Buryanov Y.I., Shevchuk T.V. (2004). Hypermethylation of 5'-region of the human calcitonin gene in leukemias: structural features and diagnostic significance. Biochemistry Mosc. 69 (3), 340–9 [+]

    Methylation of the 5'-region of the calcitonin gene was investigated in bone marrow and peripheral blood cells of 27 healthy volunteers and 25 leukemic patients. In all patients suffering from various forms of myeloid and lymphoid leukemia, hypermethylation of CpG sequences was observed in this region of the calcitonin gene. Cytosine hypermethylation in the CpG sequence did not involve cytosines of adjacent CpNpG sequences (where N is any nucleoside). The 5'-region of the calcitonin gene lacked CpNpG methylation both in healthy controls and in leukemic patients; this apparently represents specific "non-alternative" type of CpG methylation in the extended DNA sequence. Methylation of the calcitonin gene was monitored in 18 leukemic patients during malignant progression and medical treatment. Hypermethylation of the calcitonin gene was not observed on long-term clinical hematological remission. In ten patients characterized by unstable (or incomplete) remission hypermethylation of the calcitonin gene persisted through the whole period of observation. In relapses, hypermethylation of the calcitonin gene appeared again and in six patients, this "molecular relapse" being registered 1-8 months before onset of clinical and laboratory signs of disease progression. The leukemia-specific hypermethylation of CpG sequences of the 5'-region of the calcitonin gene is a promising prognostic and diagnostic marker of leukemias and might be useful for monitoring of this disease.

    ID:1460
  23. Clark J., Shevchuk T., Swiderski P.M., Dabur R., Crocitto L.E., Buryanov Y.I., Smith S.S. (2003). Mobility-shift analysis with microfluidics chips. BioTechniques 35 (3), 548–54 [+]

    Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

    ID:1461
  24. Rukavtsova E.B., Zolova O.E., Burianova N.I.a., Borisova V.N., Bykov V.A., Burianov Ia.I. (2003). Analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis B virus. Genetika 39 (1), 51–6 [+]

    The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBS antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HbsAg gene under a single 35 S promoter was 0.0001-0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002-0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants.

    ID:1449
  25. Poroĭko V.A., Rukavtsova E.B., Orlova I.V., Burianov Ia.I. (2000). Phenotypic changes in transgenic tobacco plants with an antisense form of the hmg1 gene. Genetika 36 (9), 1200–5 [+]

    Tobacco plants were genetically transformed with the Arabidopsis thaliana heterologous hmg1 gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme involved in the metabolism of terpenoid compounds. The hmg1 gene was inserted under the control of the 35S RNA double promoter from the cauliflower mosaic virus (CaMV 35S) both in direct and reverse orientation relative to the promoter. DNA analysis by polymerase chain reaction (PCR) and Southern blotting confirmed the transgenic nature of the tobacco plants obtained. DNA-RNA hybridization revealed expression of the hmg1 gene in these tobacco plants. The plants transformed with the antisense copy of the hmg1 gene differed from the control plants in delayed development and in flower color and shape.

    ID:1450
  26. Buryanov Y.I., Zakharchenko N.S., Shevchuk T.V., Bogdarina I.G. (1995). Effect of the M-EcoRII methyltransferase-encoding gene on the phenotype of Nicotiana tabacum transgenic cells. Gene 157 (1-2), 283–7 [+]

    The EcoRII DNA methyltransferase (M-EcoRII; MTase) modifies a cytosine in the DNA sequence CCWGG which contains a CNG methylation motif characteristic of plant DNA. The gene (ecoRIIM) encoding this MTase has been cloned into the T-DNA of the wild-type Agrobacterium Ti-plasmid pTiC58 downstream from the plant expression nopaline synthase-encoding gene promoter. Nicotiana tabacum cells have been transformed with Agrobacterium tumefaciens harbouring this recombinant Ti-plasmid. The primary transformed tabacco tissue line has given rise to novel stable lines which are morphologically distinctive. Southern hybridization analysis of all transformed tissue lines has shown the presence, in each of them, of ecoRIIM. The tissue studied differed in morphology in callus culture, dependence on phytohormones and the ability to synthesize nopaline.

    ID:1464

Yaroslav Buryanov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. ФИБХ, office. Переход/439
  • Phone: +7(4967)73-09-21#3228
  • E-mail: buryanov@bibch.ru

Biological activity of water extract of cecropin P1-expressing Kalanchoe plants (2017-11-27)

Water extract of constructed Kalanchoe pinnata plants expressing antimicrobial peptide cecropin P1 (cecP1) has been analyzed in the experiments on plants and animals. The extract has effectively induced rhizogenesis on callus culture of Mesembryanthemum crystallinum plants, difficult object for differentiation in vitro. High wound healing (Fig. 1) and and microbiocide activity of the cecP1-plant extract, better than efficiency of Cefazolin antibiotic against Staphylococcus aureus and Pseudomonas aeruginosa, causative agents of purulent infections (Fig. 2), and efficiency of Clotrimazolum antifungal drug against highly virulenr clinical isolate Candida albicans were found. These data demonstrate good prospects for use cecP1-kalanchoe plants in experimental biology and pharmaceutics.

Publications

  1. Захарченко Н.С., Фурс О.В., Пиголева С.В., Шевчук Т.В., Дьяченко О.В., Бурьянов Я.И. (2018). Биологическая активность экстрактов цекропин Р1-синтезирующих растений каланхоэ: перспективы для фармакологии. Физиология растений 65 (1), 94–100 [+]

     

    Разработан адаптированный для фармацевтики метод получения водного экстракта из трансгенных растений каланхоэ перистого (Kalanchoe pinnata L.), экспрессирующих ген антимикробного пептида цекропина Р1. Хлороформ является эффективным консервантом для водного экстракта каланхоэ. Показано, что антибиотические свойства экстракта сохраняются при его кипячении в течение часа. Кроме того, продолжительное сохранение антимикробной активности в экстракте показано методом “ускоренного старения”. Экстракт из трасгенных растений, добавленный в среду для культивирования, обеспечивает более интенсивный рост и усиливает ризогенез каллусов растений хрустальной травки (Mesembryanthemum crystallinum). Полученные результаты указывают на повышенную биологическую активность экстракта растений каланхоэ, продуцирующих антимикробный пептид цекропин Р1, и перспективность его использования в фармакологии.

    ID:1958
  2. Lebedeva A.A., Zakharchenko N.S., Trubnikova E.V., Medvedeva O.A., Kuznetsova T.V., Masgutova G.A., Zylkova M.V., Buryanov Y.I., Belous A.S. (2017). Bactericide, Immunomodulating, and Wound Healing Properties of Transgenic Kalanchoe pinnata Synergize with Antimicrobial Peptide Cecropin P1 In Vivo. J Immunol Res 2017, 4645701 [+]

    Procedure of manufacturing K. pinnata water extracts containing cecropin P1 (CecP1) from the formerly described transgenic plants is established. It included incubation of leaves at +4°C for 7 days, mechanical homogenization of leaves using water as extraction solvent, and heating at +70°C for inactivating plant enzymes. Yield of CecP1 (after heating and sterilizing filtration) was 0.3% of total protein in the extract. The water extract of K. pinnata + CecP1 exhibits favorable effect on healing of wounds infected with S. aureus (equal to Cefazolin) and with a combination of S. aureus with P. aeruginosa (better than Cefazolin). Wild-type K. pinnata extract exhibited evident microbicide activity against S. aureus with P. aeruginosa but it was substantially strengthened in K. pinnata + CecP1 extract. K. pinnata extracts (both wild-type and transgenic) did not exhibit general toxicity and accelerated wound recovery. Due to immunomodulating activity, wild-type K. pinnata extract accelerated granulation of the wound bed and marginal epithelialization even better than K. pinnata + CecP1 extract. Immunomodulating and microbicide activity of K. pinnata synergizes with microbicide activity of CecP1 accelerating elimination of bacteria.

    ID:1956
  3. Zakharchenko N.S., Belous A.S., Biryukova Y.K., Medvedeva O.A., Belyakova A.V., Masgutova G.A., Trubnikova E.V., Buryanov Y.I., Lebedeva A.A. (2017). Immunomodulating and Revascularizing Activity of Kalanchoe pinnata Synergize with Fungicide Activity of Biogenic Peptide Cecropin P1. J Immunol Res 2017, 3940743 [+]

    Previously transgenic Kalanchoe pinnata plants producing an antimicrobial peptide cecropin P1 (CecP1) have been reported. Now we report biological testing K. pinnata extracts containing CecP1 as a candidate drug for treatment of wounds infected with Candida albicans. The drug constitutes the whole juice from K. pinnata leaves (not ethanol extract) sterilized with nanofiltration. A microbicide activity of CecP1 against an animal fungal pathogen in vivo was demonstrated for the first time. However, a favorable therapeutic effect of the transgenic K. pinnata extract was attributed to a synergism between the fungicide activity of CecP1 and wound healing (antiscar), revascularizing, and immunomodulating effect of natural biologically active components of K. pinnata. A commercial fungicide preparation clotrimazole eliminated C. albicans cells within infected wounds in rats with efficiency comparable to CecP1-enriched K. pinnata extract. But in contrast to K. pinnata extract, clotrimazole did not exhibit neither wound healing activity nor remodeling of the scar matrix. Taken together, our results allow assumption that CecP1-enriched K. pinnata extracts should be considered as a candidate drug for treatment of dermatomycoses, wounds infected with fungi, and bedsores.

    ID:1957

Obtaining of plants with increased expression of HBsAg as edible vaccine against hepatitis B (2016-03-18)

The new expression system created for increased synthesis of proteins in plants under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) containing four enhancer sequences CaMV 35S and nontranslated leader sequence Ω RNA of tobacco mosaic virus. The immunogenicity of HBsAg synthesized by potato tubers was shown in experiments on mice. Increased synthesis of target proteins allows the use of transgenic plants as edible vaccine against the hepatitis B without additional injections of recombinant vaccine.

Publications

  1. Rukavtsova E.B., Rudenko N.V., Puchko E.N., Zakharchenko N.S., Buryanov Y.I. (2015). Study of the immunogenicity of hepatitis B surface antigen synthesized in transgenic potato plants with increased biosafety. J. Biotechnol. 203, 84–8 [+]

    Oral immunogenicity of the hepatitis B surface antigen (HBsAg) synthesized in the tubers of marker-free potato plantshas been demonstrated. Experiments were performed in the two groups of outbred NMRI mice. At the beginning of investigations, the mice of experimental group were fed the tubers of transgenic potato synthesizing the HBsAg three times. The mice of control group were fed nontransgenic potato. Intraperitoneal injection of the commercial vaccine against hepatitis B (0.5μg/mouse) was made on day 71 of the experiment. Enzyme-linked immunoassay (ELISA) of the serum of immunized animals showed an increase in the level of HBsAg antibodies significantly above the protective value, which was maintained for 1 year after the immunization. In 1 year, the experimental group of mice underwent additional oral immunization with HBsAg-containing potato tubers. As a result, the level of antibodies against the HBsAg increased and remained at a high protective level for several months. The findings show the possibility of usingtransgenic plants as a substance for obtaining a safe edible vaccine against hepatitis B.

     

    ID:1436
  2. Захарченко Н.С., Лебедева А.А., Фурс О.В., Рукавцова Е.Б., Шевчук Т.В., Дьяченко О.В., Бурьянов Я.И. (2015). Новая экспрессионная система для повышенного синтеза антимикробного пептида цекропина Р1 в растениях. Физиология растений 62 (4), 571–578 ID:1438