Transformação genética de plantas de fumo (Nicotiana tabacum var. Xanthi) com a seqüência CV1887 de Chromobacterium violaceum

Abstract

The genetic transformation of plants represents today an important tool to investigate the function of genes of diverse origins like plants, fungi, virus, nematodes and bacteria. Those that come from bacteria are the most representative ones. Within this context, sequences coding to domains containing YD (tyrosine - asparatate) repetitions, that have similarities with nematicide proteins, were detected in the Chromobacterium violaceum strain ATCC 12472 genome. Among these sequences, the ORF CV1887 (4.155 bp) was selected for cloning and expression in the heterologous system, in the attempt to validate its activity determined in silico in the C. violaceum genome's annotation. The experimental strategy consisted in cloning the complete (4.155 bp) and the partial sequence (2.642 bp) of the ORF in the binary vector pBI121. The recombinant vectors w ere introduced in Agrobacterium tumefaciens strain LBA4404 cells by electroporation. By utilizing agroinfection system, leaves segments of Nicotiana tabacum var. Xanthi were transformed and used as propagles for regeneration of the firsts transformants. Th e confirmation of the genetic transformation was achieved by PCR with the genomic DNA extracted from of the selected clones, followed of a PCR reaction. The visualization of bands in the agarose gel electrophoresis showed that from the 19 clones with the p artial sequence cv1887 that were selected, 84% showed bands with approximate size of 2.642 bp, and from the 13 clones with the complete sequence CV1887 that were selected, 78% showed bands with approximate size of 4.155 bp. For the expression analysis, the following were selected: three transformed clones with partial sequence CV1887, two transformed clones with complete sequence CV1887, three clones transformed with the reporter gene gus, which encodes for the enzyme b - glucoronidase, and two control clone s of non - transformed plants. The RNA from the selected clones was used in a RT - PCR reaction for cDNA synthesis and amplification of the corresponding sequences. The products of the amplification were analyzed in an agarose gel electrophoresis, showing the presence of bands with 2.642 bp for the three clones transformed with partial sequence CV1887 and 1.812 bp for the three clones transformed with gus and it is confirmed the presence of these sequences in the transformed cells. It was not confirmed the pres ence in transformed clones, the complete sequence CV1887.