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| Author(s): |
Francisco Cláudio da Conceição Lopes
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Piracicaba. |
| Institution: | Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) |
| Defense date: | 2002-07-03 |
| Advisor: | Marcio de Castro Silva Filho |
| Abstract | |
Proteinase inhibitors have been broadly employed in plant transformation aiming insect pest control. Here, we report a detailed study of the digestive proteinase activities present in the midgut of Sphenophorus levis (Coleoptera: Curculionidae) larvae, an important pest of sugarcane. It was observed a major activity of cysteine proteinases and a minor level of serine proteinases. These results led us to introduce into the sugarcane genome a new cysteine proteinase inhibitor called Chagasin. This gene was isolated from Trypanosoma cruzi and its biochemical characterization revealed that the gene product is a strong cysteine proteinase inhibitor. Enzymatic assays were performed using the recombinant form of this protein wich displayed potent inhibitory activity against the cysteine proteinases of S. levis larvae. Gene constructs carring the sequence encoding Chagasin and the reporter gene NPTII were used to transform embriogenic calli of sugarcane via biobalistic. Plants were selected from medium containing Geneticin and were further characterized at the molecular level by PCR, RT-PCR and Western Blotting. Analyses of several transgenic plants confirmed the integration and expression of Chagasin into the sugarcane. Assays using protein extracts of transgenic plants were performed to address a putative inhibitory effect of Chagasin onto the cysteine proteinases of S. levis. The results showed that here was no inhibitory effect of the sugarcane protein leaf extract on the cysteine proteinase activity of the insect larvae. This observation is probably due to the low expression level observed in the transgenic plants. (AU) | |
| FAPESP's process: | 00/02969-9 - Production and characterization of transgenic sugarcane plants expressing chagasin: effects on the Sphenophorus insect and on the sugarcane mosaic virus |
| Grantee: | Francisco Claudio da Conceicao Lopes |
| Support Opportunities: | Scholarships in Brazil - Master |