Prospecção do gene da Calmodulina em plantas

Abstract

Sugarcane is one of the most important producing monocultures in Brazil as a source of first and second-generation renewable biofuel, as well as sugar and other derivatives. The conditions of abiotic and water stress and high salinity of the soil for a region of the Northeast are unfavorable to the cultivation of plants and vegetables early.. This has a negative impact for sugarcane culture as it may reduce the production of alcohol and sugar. Molecular studies for the flowering pathway has an important impact for this culture. In this work, the aim of this work was to understand the possible role for ScCAM sequence (Calmodulin sequence in sugarcane). This sequence was previously identified related to flowering process. In order to understand the possible association of this sequence to flowering process in sugarcane it was used different approaches for this work. It was used bioinformatics, protein-protein interaction (two-hybrid assays), analysis of transgenic plants Nicotiana tabacum with the overexpression cassette in antisense orientation and histological analysis from apical meristem from sugarcane plants. First, bioinformatics analyzes allowed us to verify the conservation of the nucleotide sequence by comparing the ScCAM sequence with the sequences of AtCAM (Arabidospis thaliana), which presented 99% identity. A phylogenetic tree was done using the coding nucleotide sequences (CDS). It was observed a high conservation of calmodulin in plants. Based on these results, a 3D modeling from the ScCAM sequence was done. This model was overlapped to the AtCAM 3D model for the 4AQR crystal. This overlapped showed the EF-hands motifs and the entire protein structure. This data allowed us to propose that ScCAM may be a functional protein. After that, an interaction network was done using the AtCAM7 sequence (from Arabidopsis thaliana) using Cytoscape. The Clusters obtained were enriched and it showed proteins that may be interacting as flowering induction as well as it had a role in the oxidation/reduction homeostasis. The two hybrids assays identified some clones and tow of these clones had homology to A. thaliana ribosomal proteins L31-1 and L19-1 (Uniprot access: Q9SLL7). These results suggest that the ScCAM protein may interact with ribosomal proteins. The transgenic plants having the overexpression cassete in antisense (ScCAMAS) orientation showed an increase in the germinated potential of the transgenic seeds ScCAM-AS, an increase of inflorescence branches number and fruit. Furthermore, it was observed an increase of root hair when compared to transgenic plants having the empty vector (control) or wild-type plants. For leaves, it was not observed any changes at morphometric parameters. The only modification that was observed for these plants were the height, inflorescence and fruits. In parallel, it was also analyzed the shoot apical meristem from sugarcane plant at production field using early and late-flowering cultivar. This analysis showed that the flowering induction may be happen 30-60 days before for early flowering cultivar. Similar results were observed for the transgenic plants. In this way, the results obtained here reinforce the hypothesis that the ScCAM sequence may be associated to the flowering process as well as stress signaling pathway by modification from physiological processes, then it may be remodeling apical and root meristems. The proteins identified in the interaction network indicated that AtCAM may act in pathways of stress signaling by altering physiological processes in the remodeling of meristematic tissues and roots by interactions between Ca+2 + CAM and actin modulating time of growth, development and flowering. The results obtained with transgenics plants and the data with the double hybrid suggest that ScCAM may act together with the ribosomal 60S protein as a floral inducer through the activation or inhibition from genes belonging to the flowering pathway. As well, it may potentiating the increase of biomass.