Laboratory of Plant Biotechnology

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Taras Shevchuk

Obtaining and Analysis of Marker-free Oil Plants Camelina sativa (L.) Expressing Gene for Antimicrobial Peptide Cecropin P1

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selection genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral, antistress, plant-protective system.

Diversity of rhodopsins in cultivated bacteria of the family Geodermatophilaceae associated with non-aquatic environments

A large number of different rhodopsins were found in cultivated bacteria isolated from hot and arid ecological niches. A total of 31 rhodopsin genes were identified in 51 analyzed genomes of strains belonging to the family Geodermatophilaceae. Overall, 88% of the strains harbouring rhodopsins are isolated from non-aquatic environments. It was found that 82% of strains belonging to the genus Geodermatophilus have at least one gene as compared to 38% of strains of other genera which contain rhodopsins. Analysis of key amino acids revealed two types of the studied proteins: DTE type (putative proton pump) and NDQ type (putative sodium pump). Proton pumps were divided into two subtypes (DTEW and DTEF) according to phylogenetic analysis and the presence of highly conserved tryptophan or phenylalanine at position 182. Among all studied rhodopsins DTEF subtype is the most unique one, identified only in this family.

The picture shows phylogenetic tree based on rhodopsin protein sequences found in bacteria of the family Geodermatophilaceae.

Resistance of bombinin-expressing plants to phytopathogens

Transgenic plants expressing artificial gene of an antimicrobial peptide bombinin have been produced and investigated. This plants were resistant to the Erwinia carotovora bacteria and Rhizoctonia solani fungi phytopathogens. Such plants expressing the small antimicrobial peptide gene are promising for new approach in plant protection agriculture technology.

Biological activity of water extract of cecropin P1-expressing Kalanchoe plants

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.

Obtaining of plants with increased expression of HBsAg as edible vaccine against hepatitis B

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.