RESUMEN
While the authors have previously developed a method of pistil filament treatment with Agrobacterium cells during blossoming for the transformation of maize generative cells, the mechanism for bacterial T-DNA penetration into the embryo sac remained unknown. This article analyzes the possibility of agrobacterial penetration into the maize embryo via pollen tubes. Microbiological, PCR, and GUS techniques were used to confirm that agrobacteria could spread for up to 20 cm from the sie of inoculation and were detected in maize embryo tissues as aerly as 24 h after inoculation, while they were not revealed after 5-13 days.
Asunto(s)
Agrobacterium/crecimiento & desarrollo , Proliferación Celular/fisiología , Flores/microbiología , Viabilidad Microbiana , Zea mays/microbiología , Agrobacterium/ultraestructuraRESUMEN
The bacteriophage ΦAb-Sp7 was isolated from the cells of the Azospirillum brasilense Sp7. The morphology, size of the gram-negative colonies, and range of lytic activity against other strains and species of the genus Azospirillum was tested. The isolated phage DNA was examined using electrophoretic and restriction analysis, and the size of the genome were established. The electron microscopy. resuIts show that the phage (capsid) has a strand-like form. The electron microscopy study of the bacteriophage ΦAb-Sp7 adsorption on the A. brasilense Sp7 bacterial surface was performed.
Asunto(s)
Azospirillum brasilense/virología , Bacteriófagos/genética , ADN Viral/química , Genoma Viral , Adsorción , Azospirillum brasilense/química , Azospirillum brasilense/ultraestructura , Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/ultraestructura , Cápside/ultraestructura , Enzimas de Restricción del ADN/química , Tamaño del Genoma , Lisogenia , Mapeo RestrictivoRESUMEN
Abstract-A transfer DNA (T-DNA) carrying the marker gene nptII was detected in the genomes of diploid and haploid maize plants obtained after the treatment of pistil filaments with a suspension of Agrobacterium during artificial pollination. PCR analysis of total DNA isolated from 155 canamycin-resistant diploid F1 seedlings revealed T-DNA insertions in the genomes of 111 plants (32.7% of the total number of analyzed seeds). The example of matroclinal haploids was used to demonstrate that T-DNA may be transported to the egg cell by the growing pollen tube (PT). Twelve out of 16 analyzed haploid plants contained the T-DNA insertion. The possible mechanism of the transfer of the Agrobacterium T-DNA to the maize genome during pollination is discussed.
Asunto(s)
ADN Bacteriano/genética , Plantas Modificadas Genéticamente/genética , Transformación Genética , Zea mays/genética , Diploidia , Flores/citología , Flores/genética , Haploidia , Plantas Modificadas Genéticamente/citología , Rhizobium , Zea mays/citologíaRESUMEN
The nucleotide sequences of mitochondrial genome fragments containing the genes coding for cytochrome oxidase subunit III (COIII) and 12S ribosomal RNA of common European viper and Nikolsky's viper from various habitats (Saratov, Samara, and Penza oblasts; Chuvash Republic; and the Republic of Mordovia) were determined. According to the sequencing data, all samples clustered into two groups except for a number of individuals carrying single mutations in the genes in question. One group comprised V. nikolskii from Saratov oblast and the other, V. berus from the Chuvash Republic, Republic of Mordovia, and Samara and Penza oblasts. These results comply with the available data on the karyotypes of the studied vipers of this region. Further genetic studies of V. nikolskii and V. berus from various parts of this area are necessary.
Asunto(s)
ADN Mitocondrial/genética , ADN Ribosómico/genética , Complejo IV de Transporte de Electrones/genética , Genes de ARNr/genética , ARN Ribosómico/genética , Viperidae/genética , Animales , Femenino , Masculino , Federación de RusiaRESUMEN
Integration of T-DNA into the maize genome as a result of treatment of silks with Agrobacterium cells, containing activated vir genes, was demonstrated. In planta treatment of maize (Zea mays L) was performed during flowering in field. Cell suspension of Agrobacterium tumefaiciens strain GV3101(pTd33), carrying activated vir genes, was applied onto the previously isolated silks, which were afterwards pollinated with the pollen of the same cultivar. Integration of T-DNA into maize genome was confirmed by PCR (the nptII and gus reporter genes) and hystochemical staining of the seedling tissues, obtained from the transformed seeds. Amplification of the nptII gene showed the presence of about 60.3% of PCR-positive plants out of the total number of kanamycin-resistant seedlings examined, or 6.8% of the total of number of seedlings.
Asunto(s)
Agrobacterium tumefaciens/genética , ADN Bacteriano/genética , Técnicas de Transferencia de Gen , Transformación Genética , Zea mays/genética , ADN de Plantas/análisis , Marcadores Genéticos , Vectores Genéticos/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
The scFv miniantibodies to the recombinant protein VirE2 from Agrobacterium tumefaciens were obtained by the method of phage display. The miniantibodies were purified and tested using timmunodot method for binding to a recombinant protein from Escherichia coli and to the native protein VirE2 from A. tumefaciens. The functional activity of the miniantibodies was comparable to the activity of mouse polyclonal antibodies against the VirE2 protein.
Asunto(s)
Agrobacterium tumefaciens/inmunología , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Proteínas de Unión al ADN/inmunología , Canales Iónicos/inmunología , Proteínas Recombinantes/inmunología , Agrobacterium tumefaciens/química , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/aislamiento & purificación , Especificidad de Anticuerpos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/metabolismo , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Canales Iónicos/genética , Canales Iónicos/aislamiento & purificación , Biblioteca de Péptidos , Proteínas Recombinantes/biosíntesisRESUMEN
Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid, T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein with six histidine residues at the N-end was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2 which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.
Asunto(s)
Agrobacterium tumefaciens/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Canales Iónicos/aislamiento & purificación , Agrobacterium tumefaciens/genética , Animales , Anticuerpos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Clonación Molecular , ADN de Cadena Simple/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Electroforesis en Gel de Agar , Canales Iónicos/genética , Canales Iónicos/inmunología , Biblioteca de Péptidos , Plásmidos , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , VirulenciaRESUMEN
Exocellular structures containing VirB2 proteins were, for the first time, localized on the surface of Agrobacterium by transmission electron microscopy. Using colloidal gold (CG)-labeled VirB2-specific antibodies, it was shown that VirB2 proteins enter into the composition of short surface pili, which emerge at the poles of acetosyringone (AS)-inducedAgrobacterium cells. However, cells of the Ti plasmidless A. tumefaciens strain UBAPF-2 and cells not induced with AS were incapable of pilus synthesis. In suspension, mating Agrobacterium cells were connected together by short thick bridges. It was found that these bridges did not include as part of their structure CG-labeled VirB1 and VirB2 proteins. We did not find the tetracycline-resistant transconjugants after mating of A. tumefaciens donor cells harboring binary systems with plasmid-free A. tumefaciens GM-I9023 in vir-induced and vir-uninduced conditions. However, the same strains can transfer pSUP106 plasmid via a vir-dependent way. We found that activated vir genes slightly stimulate pTd33 plasmid transfer via a tra-dependent pathway to plasmid-free strain UBAPF-2. It seems, that vir-induced T-DNA/plasmid DNA transfer machinery is not essential for the conjugation process between agrobacterial cells but may participate in this process.
Asunto(s)
Agrobacterium tumefaciens/genética , Proteínas Bacterianas/metabolismo , Conjugación Genética , Fimbrias Bacterianas/metabolismo , Plásmidos , Factores de Virulencia , Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/ultraestructura , Proteínas Bacterianas/genética , Conjugación Genética/genética , Fimbrias Bacterianas/ultraestructura , Técnicas de Transferencia de Gen , Microscopía Electrónica , Plásmidos/genéticaRESUMEN
The process of the transfer of the Ti-plasmid vector pGV3850 with the plasmid pBR322 inserted into the T-DNA region from Agrobacterium tumefaciens to a non-plasmid strain of Escherichia coli was studied. The transferred Ti-plasmid was found to be exposed to deletions and formed a wide range of derivatives with a size ranging from 3-4 kb to 50 kb, maintained in E. coli due to ColE1-replicon. The Ti-plasmid is also inserted into the chromosome of the recipient bacterium with at least a 100-fold lower frequency than the formation of deletional derivatives. It was shown that the induction of vir genes controlling the transfer of T-DNA into plants has no appreciable effect on the efficiency of obtaining transconjugates in mating with E. coli. The deletion of the genetic material of megaplasmids with the inserted functional site OriV ColE1, as a result of the conjugative transfer from cells of different bacteria to the cells of E. coli, was proposed for molecular cloning.