Caracterização de plantas de Nicotiana tabacum transformadas com sequência codificadora de uma quitinase de feijão-de-corda (Vigna unguiculata).

Abstract

The chitinases enzymes are capable of promoting the cleavage of the links between the monomers of N-acetylglucosamine. In the context, Nicotiana tabacum plants transformed with a VuChiI sequence encoding cowpea bean chitinase (Vigna unguiculata), were used as a model to perform characterization studies and protein activity. In the present work, we report the analysis of transcripts for VuChiI, the segregation analysis of transformed plants, chitinase activity of plant extracts and the fractionation of protein extract. That way, transgenic plants of Nicotiana tabacum containing a chitinase from V. unguiculata were evaluated. The expression of the gene in three generations of transformed plants was confirmed by RT-PCR. Two generation of transgenic plants produced by autofertilization of parents containing the insert was tested for the stability of the inheritance of the transgene. In both descendants the pattern of segregation did not differ from the expected Mendelian proportions. The production of chitinase in protein extracts of leaves was analyzed by colorimetric assay using colloidal chitin as substrate. Enzymatic activity was detected, although its levels had changed in different transformed plants. Chromatography on chitin column was carried out and the fractionation of the extract showed two peaks. The expression of the gene was confirmed, as well as the enzymatic activity of the protein.