Al. Kh. Baymiev

The Phylogeny of Triticum L. and Aegilops L. Inferred from Comparative Analysis of Nucleotide Sequences in rDNA Promoter Regions

The process of accumulation of knowledge on wheat and related wild species during the 20th century is briefly reviewed with special reference to the evidence of the recent years on evolution of polyploid wheats and the role of diploid species. The latter serve as potential donors of the genomes, detection of which is particularly important because of the continuing speciation in the tribe Triticeae and artificial development of synthetic forms. The arguments in favor of the donor role for various diploid wheat and aegilops species from the section Sitopsis are compared. It is stated that in the formation of the both lines of polyploid wheats turgidum–aestivumand timopheevi,diploid Aegilops speltoides acted as a maternal form. In addition to cytoplasmic genomes, this aegilops species introduced into them also the B and G nuclear subgenomes. A comparison of nucleotide sequences in the variable part of the promoter of evolutionary conserved rRNA genes in polyploid wheats with their counterparts in diploid wheats and aegilops species confirmed the accepted wheat phylogenies.

Polymorphism of lectin genes in Lathyrus plants

The carbohydrate-binding sequences of the lectin genes from spring vetchling Lathyrus vernus (L.) Bernh., marsh vetchling L. palustris (L.), and Gmelin’s vetchling L. gmelinii (Fitsch) (Fabaceae) were determined. Computer-aided analysis revealed substantial differences between nucleotide and predicted amino acid sequences of the lectin gene regions examined in each of the three vetchling species tested. In the phylogenetic trees based on sequence similarity of carbohydrate-biding regions of legume lectins, the sequences examined formed a compact cluster with the lectin genes of the plants belonging to the tribe Fabeae. In each plant, L. vernus, L. palustris, and L. gmelinii, three different lectin-encoding genes were detected. Most of the substitutions were identified within the gene sequence responsible for coding the carbohydrate-binding protein regions. This finding may explain different affinity of these lectins to different carbohydrates, and as a consequence, can affect the plant host specificity upon development of symbiosis with rhizobium bacteria.

Genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine Lathyrus vernus (L.) Bernh.

The genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine Lathyrus vernus (L.) Bernh. (Fabaceae) growing in various regions of the Republic of Bashkortostan were studied. RAPD analysis revealed a high degree of polymorphism of the DNA of the isolated strains giving evidence of the heterogeneity of the microorganisms in question. The study of the phylogeny of microsymbionts based on comparative analysis of the nucleotide sequences of 16S rRNA genes showed that the bacteria isolated from the plant nodules of L. vernus growing on the territory of Ufa and Beloretsk raions belonged to the species Rhizobium leguminosarum, whereas the microsymbionts of L. vernus growing on the territory of Tatyshly raion belonged to the species Rhizobium tropici,@ except for several strains of Rhizobium leguminosarum

Analysis of symbiotic genes of leguminous root nodule bacteria grown in the southern urals

Bacterial strains isolated from the nodules, tissues, and root surface of wild legumes growing in the Southern Urals related to the tribes Galegeae, Hedysareae, Genisteae, Trifolieae, and Loteae were examined for the presence in their genomes of symbiotic (sym) genes. It was found that the sym-genes are present in microorganisms isolated only from the nodules of the analyzed plants (sym+ strains). Phylogenetic analysis of sym+ strains on the basis of a comparative analysis of 16S rRNA gene sequences showed that sym+ strains belong to five families of nodule bacteria: Mesorhizobium, Bradyrhizobium, Sinorhizobium, Rhizobium, and Phyllobacterium. A study the phylogeny of the sym-genes showed that the nodule bacteria of leguminous plants of the Southern Urals at the genus level are mainly characterized by a parallel evolution of symbiotic genes and the 16S rRNA gene. Thus, cases of horizontal transfer of sym genes, which sometimes leads to the formation of certain types of atypical rhizobial strains of leguminous plants, are detected in nodule bacteria populations.

Role of Bacterial Adhesin RAPA1 in Formation of Efficient Symbiosis of Rhizobium leguminosarum with Bean Plants

Bacterial adhesins are the proteins responsible for attachment of plant growth-promoting rhizobacteria (PGPR) to plant roots, are involved in formation of stable associative symbiosis. In the present work, enhanced expression of the adhesin gene rapA1 in Rhizobium leguminosarum PVu5 was shown to improve the efficiency of root nodulation on bean roots inoculated with the modified strain. The gene rapA1 was cloned into the pJN105Turbo plasmid, this construct was used for transformation of R. leguminosarum PVu5, bean plants were inoculated by this transgenic strain, and efficiency of root nodule formation was determined. In the plants treated with rapA1-transgenic rhizobia, the number of root nodules was on average two times higher than in the plants inoculated with the original strain. Aggregation of R. leguminosarum was achieved when the gene rapA1 expression was enhanced either in rhizobia or in the co-cultured modified strain E. coli pJN105TurboRapA1.

Genetic description of root nodule bacteria of Lathyrus species growing on the territory of the Republic of Bashkortostan

The genetic diversity and phylogeny of rhizobia isolated from nodules of nine wild-growing Lathyrus L. species (Fabaceae) growing in the Republic of Bashkortostan were studied. It is shown that, for the given plants, a large variety of heterogeneous strains of root nodule bacteria is characteristic. Nevertheless, it is revealed that the majority of them, in terms of phylogenetics, are closely related to Rhizobium leguminosarum. However, some plant species are found also: nodule bacteria, which were earlier considered unusual for Lathyrus. Thus, L. vernus L. Bernh. and L. sylvestris L. are found to have a root nodule bacteria close to R. tropici, L. palustris L. Agrobacterium sp., and L. gmelinii Fritsch, all bacteria isolated by us from root nodules by the sequence of genes of 16S rRNA, are closely related to Phyllobacterium myrsinacearum.

Preparation of fluorescent labeled nodule bacteria strains of wild legumes for their detection in vivo and in vitro

A series of expression vectors containing TurboGFP and TurboRFP genes of fluorescent proteins under the control of the T5 phage constitutive promoter was created for a vital staining of nodule bacteria. These vectors were either obtained using the broad host range pBBRI replicon for labeling of strains, where a marker gene was expressed from a transformed plasmid, or they were prepared using the pRL765 gfp plasmid for labeling of strains via the introduction of genes of fluorescent proteins into the bacterial chromosome. Transformation was shown to be the most convenient method of transfer of constructions into cells of nodule bacteria, as there exists the possibility of spontaneous plasmid mobilization and, consequently, its transition from cells of labeled strains into other soil bacteria if the mob locus is present in vectors needed for conjugation. Fluorescent labeled strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., and Agrobacterium sp. were prepared using the obtained vector constructions. The suitability of the obtained strains for both in vivo and in vitro experiments was demonstrated.

Inverse PCR-based site-directed mutagenesis of nucleotide sequences coding for carbohydrate-binding fragments of legume lectins

A new method of site-directed mutagenesis was developed to allow manipulation with extended plasmid-cloned gene fragments irrespective of their position and the presence of restriction sites. The method was used to obtain chimeric constructs encoding a Pisum sativum lectin with the native carbohydrate-binding region replaced by its counterpart from other legumes. The method can be used in plasmid construction to clone a coding gene fragment under the control of a promoter in a certain reading frame.

Quantitative Analysis of the Microbiota of Periodontal Pockets and Saliva by Real-Time PCR before and after Treatment of Periodontitis

Work has been done on standardization of detection and quantification of periodontopathogenic microorganisms in the clinical material (contents of periodontal pockets and saliva) using real-time PCR. To optimize the conditions for the analysis, a method for obtaining clinical samples of known volume was developed and a calibration sample to obtain reliable results in the diagnosis of periodontitis was designed.

Genetic markers for search of rhizobia based on symbiotic genes

The possible application of rhizobial symbiotic genes as markers for the search and primary identification of rhizobia from temperate-zone legumes was studied. It was shown that conservative sym genes nifH and nifD could be used as markers for rapid search of rhizobia among the analyzed isolates, while more variable genes nifK and nodC could be used for their primary identification. Efficiency of the proposed method was shown in analysis of bacterial isolates obtained from Onobrychis arenaria and Astragalus cicer root nodules.