Lavina A.M., Khakimova L.R., Matniyazov R.T., Vershinina Z.R., Baymiev A.Kh.
THE OBTAINING OF THE RECOMBINANT STRAINS OF RHIZOBIA CARRYING AN EXTRA COPY OF GENES PSSA AND ROSR LABELED WITH FLUORESCENT PROTEIN GFP ^{[№ 9 ' 2017]}
The establishment of specific symbiotic relationships between rhizobia and leguminous plants is a complex process that requires the exchange of signaling intermediary molecules between both partners. The flavonoids, secreted by the roots of leguminous plants, induce rhizobium expression of nodule-forming genes — Nod-factors. Synthesis of Nod factors is necessary, but not sufficient for the morphogenesis of effective nitrogen fixing nodules. Since no less important factor in the formation of nodules is the synthesis of rhizobacteria exopolysaccharides (EPS), which play a decisive role in the symbiotic interactions of nodule bacteria with leguminous plants. In addition, in free-living cells, rhizobia EPSs are responsible for attachment to abiotic and biotic surfaces and for the formation of biofilms that ensure the adaptation of bacteria to changing environmental conditions. Currently, the formation of biofilms by rhizobia and the relationship of this process with effective nodule formation is being intensively studied. Special attention should be paid to the rosR gene, which encodes the transcriptional regulator involved in EPS biosynthesis, as well as the pssA gene responsible for initiating the process of EPS biosynthesis in rhizobia.
To study the genes responsible for the synthesis of EPS in the context of their impact on the formation of biofilms by rhizobia, in particular in artificial symbiotic systems, where colonization of plant roots plays an important role, it is necessary to visualize the interaction of nitrogen fixing bacteria with plant roots. In view of this, the aim of this work was to obtain recombinant rhizobial strains labeled with fluorescent protein GFP by the pssA and rosR genes. To do this, a genetically engineered construct containing the fluorescent protein gene GFP was used, which serves to visualize the interaction of rhizobia with plant roots. Cloning of pssA and rosR genes into plasmid pJB658GFP, transformation of rhizobium with this construct, microscopic examination of the obtained recombinant strains was carried out.
It has been shown that recombinant rhizobial strains with an additional copy of the pssA or rosR gene are characterized by a large mismatch of the cell walls, compared to wild strains, which indicates their high production of exopolysaccharides and, as a consequence, indicates an increase in the competitiveness of the strains.