Examinando por Autor "Retamales, Julio"
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Ítem A Rapid and Efficient Method for Purifying High Quality Total RNA from Peaches (Prunus persica) for Functional Genomics Analyses(Sociedad de Biología de Chile, 2005) Meissel, Lee; Fonseca, Beatriz; Gonzalez, Susana; Baeza-Yates, Ricardo; Cambiazo, Veronica; Campos, Reinaldo; Gonzalez, Mauricio; Orellana, Ariel; Retamales, Julio; Silva, HermanPrunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries.Ítem Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)(BMC, 2010-01-18) Nilo, Ricardo; Saffie, Carlos; Lilley, Kathryn; Baeza-Yates, Ricardo; Cambiazo, Verónica; Campos-Vargas, Reinaldo; González, Mauricio; Meisel, Lee A.; Retamales, Julio; Silva, Herman; Orellana, ArielBackground: Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury.Results: The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest.Conclusions: Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest. © 2010 Nilo et al; licensee BioMed Central Ltd.