Caracterização de plantas transformadas de Urochloa brizantha sob déficit hídrico e validação de genes de referência para estresse salino

Abstract

Forage grasses, including those of the genus Urochloa sp., are of great importance for the meat and milk production chains in Brazil, however, there are still few studies aimed at improving drought tolerance, considering that with the predicted climate changes, there is a demand for resistant crops to that stress. Transgenic plants with the P5CS gene of Vigna aconitifolia under the control of the AIPC stress-induced promoter would be a suitable alternative, considering that proline plays an extremely important role in mitigating water deficit. Chapter 1 of this thesis aimed to evaluate U. brizantha plants transformed the AIPC:P5CS construct for biochemistry, physiology and molecular characterization in response to water deficit conditions. For this, the plants (control and transformed) were conducted for approximately 30 days with daily drip irrigation, close to their field capacity, and after the water deficit was imposed by water suspension until the plants reached water potential values of -3.0 to -3.5 MPa, where the content of proline, MDA, chlorophyll, leaf gas exchange, biomass and transcriptional profile of the native gene UbP5CS and the transgene VaP5CS were analyzed. The analysis of three brachiaria events transformed with the AIPC:P5CS construct revealed that the E3 event presented physiological, biochemical and molecular differences compared to the other events and the control. In addition, this event exhibited a phenotypic difference from the others. Even with these differences, there was no evidence of greater tolerance to the water deficit of the events in relation to the untransformed control. Chapter 2 refers to the selection of reference genes for salt stress in U. brizantha. Salinity is one of the most important abiotic stresses that affect plant productivity. Quantitative gene expression using real-time PCR (RT-qPCR) is a powerful tool for measuring transcriptional changes that occur in plant tissues under stress. For valid RT-qPCR analysis, normalization against the appropriate reference genes is essential for data accuracy. Despite the importance of signal grass (syn. Urochloa) as a tropical forage, there are no studies on the stability of reference genes under saline stress in this species. This study aimed to evaluate the stability of seven candidate reference genes: Actin 12; eukaryotic initiation factor 4A; elongation factor 1- α; α-5 tubulin; β-6 tubulin; ubiquitin-conjugating enzyme and glucose-6-phosphate dehydrogenase for quantitative real-time PCR assays in Urochola brizantha under saline stress. Total RNA was extracted from leaves and roots of plants grown in a hydroponic system containing 200 mM NaCl for 0, 6, 12 and 24 hrs for RT-qPCR analysis. We used the RefFinder web tool to establish a comprehensive ranking for gene stability. We identified that eIF4a, α-Tub5 and Ubico were the most suitable reference genes for leaf samples, while eIF4a, Act12 and Ubico were the most stable genes in roots. The most and least stable genes were then used to normalize the relative expression of a Na+/H+ (NHX) transporter gene. The relative quantification of this gene varied according to the internal controls (more stable genes, less stable genes), confirming the choice of reference genes.