Identificação e caracterização de componentes da via de excisão de bases (BER) em cana-de-açúcar (SACCHARUM spp.)
The productivity of any cultivar is directly related to the preservation of its genetic code, since any change in the sequence can have consequences that directly affect the development/growth of the plant. The response to DNA damage occurs through its repair through different pathways, one of wh...
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| Formato: | doctoralThesis |
| Idioma: | por |
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| Endereço do item: | https://repositorio.ufrn.br/jspui/handle/123456789/26229 |
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| Resumo: | The productivity of any cultivar is directly related to the preservation of its genetic
code, since any change in the sequence can have consequences that directly
affect the development/growth of the plant. The response to DNA damage occurs
through its repair through different pathways, one of which is the base excision
pathway (BER), whose studies on plants are still inferior in comparison to other
eukaryotic organisms. One of the obstacles in the advancement of these
researches is the complexity of the plant genome present in some cultivars of
agroeconomic importance, so that many studies use diploid models organisms.
This work proposes to fill the gap in the knowledge of the BER pathway in relation
to non-models and polyploid plants, targeting the sugarcane. The first approach
of the work was to identify components of the BER pathway in sugarcane and
compare the results obtained with the sequences of other plant organisms to
deepen the question of conservation of this pathway. The second approach was
using a sequence homologous to the AP endonuclease of Arabidosis thaliana
(AtARP) in sugarcane denominated ScARP1. This was the subject of an
enzymatic characterization test. For the first approach, we considered the results
of previous studies, where a duplication in sugarcane was verified for the AP
endonuclease sequence (ScARP1 and ScARP3), in addition to recent work on
the BER pathway review in plants. Thus, a search of the ESTs sequences
databases for sugarcane (SUCEST-FUN) was done by sequences belonging to
via BER. To verify the occurrence of duplications, the results of this search were
submitted to phylogenetic analysis and Bayesian inferences. The sequences
found were characterized as the presence of conserved domains, besides some
of them were modeled, creating hypothetical 3D models. Some of the presumed
proteins identified differed in their structure with the reference proteins of A.
thalina. Furthermore, multiple copies sequences were observed in certain plant
families over others. For the second approach, the ScARP1 protein was cloned,
expressed and purified. With this, different assays were carried out to evaluate
its enzymatic efficiency (considering temperature, enzymatic cofactors and salt
concentration), as well as the substrates that would be recognized by ScARP1.
It has been observed that the ScARP1 protein has only AP endonuclease activity.
In addition, partial complementation of ScARP1 in protein extracts of the A.
thalina arp-/- mutant was observed. With this work it was possible to identify
members of the BER pathway in sugarcane, besides characterizing them
structurally and phylogenetically, which made it possible to highlight differences
between sequences of monocotyledons and dicotyledons. Furthermore, in
relation to ScARP1, it was determined that this presents Ap endonuclease activity
essential for his performance in BER. Thus, it amplified the knowledge this
pathway in sugarcane and vegetables on organisms in general. |
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