Estabelecimento de uma cana-de-açúcar transgênica superexpressando o gene da canacistatina (CaneCPI-1), uma proteína inibidora de cisteíno-protease
Resumen
Sugarcane is a plant of great economical importance, mainly in Brazil, which is
currently the largest producer of this crop in the world. However, sugarcane farming has
suffered attacks from pests and pathogens, leading to considerable economical losses.
Therefore, a number of studies have focused on the development of more resistant
sugarcane varieties. Our laboratory has been working on a sugarcane protein (CaneCPI-
1), which is a cystein-protease inhibitor protein. Based on studies that indicate that
insects belonging to the order Coleoptera possess cystein-proteases in their mid-gut and
with the knowledge that some species of Coleoptera are sugarcane pests, a transgenic
sugarcane plant overexpressing the CaneCPI-1 gene was developed under the control of
maize ubiquitin promoter. CaneCPI-1 was fused to a His-tag to facilitate further
purification through affinity chromatography. The calli transformation was performed
through biobalistics. The transformed plants were then selected by polymerase chain
reaction. Positive plants were further selected by Semi-quantitative PCR and Western
blotting. A transformed plant expressing a His-tagged CaneCPI-1 was selected for
purification of the recombinant protein in a nickel column, enabling purification of
HISCaneCPI-1 from plant leaves in a single step. The yield was about 6mg of pure
protein per kilogram of sugarcane leaves. The HISCaneCPI-1 purified from the
transformed sugarcane demonstrated inhibitory activity on the human cysteine protease
cathepsin L. These studies demonstrate that the sugarcane can be a safe and viable
expression system for recombinant protein production, and are the first step in the
establishment of a sugarcane plant that is more resistant to pathogens.