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| Author(s): |
Ligia Hansen Arruda
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Piracicaba. |
| Institution: | Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) |
| Defense date: | 2011-05-18 |
| Examining board members: |
Marcio de Castro Silva Filho;
Pio Colepicolo Neto;
Daniel Scherer de Moura;
Goran Neshich;
Claudia Barros Monteiro Vitorello
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| Advisor: | Marcio de Castro Silva Filho |
| Abstract | |
Plants have developed different mechanisms to reduce insect attack, including defence proteins such as proteinase inhibitors (PIs). In turn, insects have evolved strategies to overcome these plant defence mechanisms, such as the hyperexpression of PI-sensitive and insensitive digestive enzymes, allowing the insect to thrive. One of the aims of this work was to identify new serine proteinases (SPs) in the gut of the fall armyworm larvae, Spodoptera frugiperda. Two new chymotrypsins and three new trypsins were identified, and together with 10 previously identified genes, the genes that encode these enzymes were subjected to real-time PCR and gene expression analysis. Between these two families of serine-proteinases the genes that encode chymotrypsins show a greater positive regulation then those encoding the trypsins. Molecular modelling studies of the chymotrypsins were carried out, and 3D models were generated using homology modelling, which were then further refined by dynamic molecular and docking analyses with 8 different Bowman-Birk type PIs. The results demonstrate which chymotrypsins possess the highest affinities to the tested inhibitors in a general and individual manner, inferred from the estimated free energies. A serine residue in very close proximity to the catalytic site was present in three of chymotrypsins investigated, which may be affecting the enzymes affinity since the residue has a reduced accessible area to the solvent when complexed to the soya PI tested. The genetic expression patterns and the degree of PI-sensitivity were also compared and no relation between the parameters was found. This suggests that the larvae of the species S. frugiperda combine different adaptive strategies like the increase in expression of its entire chymotrypsin arsenal regardless of the degree of PI-sensitivity of the enzymes. (AU) | |