Browsing by Author "Rodríguez, Hicler N."

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A simple and efficient method for high-quality total RNA isolation from oleaginous microalgae
(Plant Cell Biotechnology and Molecular Biology, 18(1-2), 15-21, 2017-01-06) Castro, Juan C.; Rodríguez, Hicler N.; Maddox, J. Dylan; Jiu, Bruce; Petterman, Joe B.; Marapara, Jorge L.; Cobos, Marianela
Microalgae are a very diverse group of microorganisms with great potential for biotechnological applications. To best exploit these organisms is necessary to expand our knowledge at its molecular levels. To realize these studies, however, it is essential to have efficient and reproducible methods for total RNA isolation. The objective of this research was to develop a method for high-quality total RNA isolation from three oleaginous microalgae species promissories for biotechnological applications. Three strains Ankistrodesmus sp., Chlorella sp., and Scenedesmus sp. were cultured on CHU-10 medium. Then, were harvested by centrifugation and total RNA was isolated with three commercial kits, one previously published method, and our improved CTAB-LiCl method. Quality and quantity of total RNA isolated were assayed with standard spectrophotometric and electrophoretic techniques. The highest quality (A260/A280: 1.95-2.02, A260/A230: 1.12-1.84) and yield (16.40-93.63 mg/g fresh weight) of total RNA from the three microalgae strains were obtained with our improved CTAB-LiCl method. In addition, the time required to complete the isolation process and the cost were relatively lowest. In conclusion, the improved CTAB-LiCl method, in contrast to frequently used commercial kits, and a previously published method, is effective and reliable to produce total RNA of high quality and quantity that is suitable for cDNA synthesis and polymerase chain reaction.
Caracterización in silico y análisis de la expresión de la subunidad alfa de la acetil-coenzima a carboxilasa heteromérica de dos microalgas
(Universidad Nacional de Colombia, 2019) Castro, Juan C.; Maddox, J. Dylan; Estela, Segundo L.; Rodríguez, Hicler N.; Casuso, María Z.; Paredes, Jae D.; Cobos, Marianela
Las microalgas son microorganismos fotosintéticos con gran potencial para abastecer las demandas energéticas mundiales. Sin embargo, los limitados conocimientos que se tienen de estos organismos, en particular a nivel molecular de los procesos metabólicos, han limitado su uso con estos propósitos. En esta investigación se ha realizado el análisis in silico de la subunidad alfa de la acetil-Coenzima A carboxilasa heteromérica (αACCasa), una enzima clave en la biosíntesis de lípidos de las microalgas Chlorella sp. y Scenedesmus sp. Asimismo, se ha medido la expresión de este gen en ambas especies cultivadas en medios deficientes de nitrógeno. Los resultados indican que la αACCasa muestra conservación estructural y funcional en ambas especies de microalgas y su mayor similitud genética con otras especies de microalgas. Asimismo, se ha mostrado que el nivel de expresión del gen se incrementa significativamente cuando las microalgas son cultivadas en ausencia de nitrógeno, lo cual se relaciona a su vez con una mayor acumulación de lípidos microalgales. En conclusión, el análisis in silico de la αACCasa de Chlorella sp. y Scenedesmus sp. presentan características estructurales, funcionales y evolutivas muy similares con otras especies de microalgas y plantas. Asimismo, el estudio revela que en ambas especies el gen se sobreexpresa cuando las microalgas son sometidas a estrés por deficiencia de nitrógeno, el cual se relaciona significativamente con la acumulación de lípidos totales en estas células.
De novo RNA-Seq analysis of the oleauginous microalgae Ankistrodesmus sp. UCP0001: Gene identification and metabolic pathways reconstruction for the biosynthesis of fatty acids and triacylglycerols
(Plant Cell Biotechnology and Molecular Biology 18(5-6): 219-230, 2017-05-15) Castro, Juan C.; Maddox, J. Dylan; Paredes, Jae D.; Rodríguez, Hicler N.; Aguilar, Carla P.; Marapara, Jorge L.; Castro, Carlos G.; Casuso, María Z.; Cobos, Marianela
Microalgae have great potential as feedstock to produce next-generation biofuels. The scarceness of genomic level information, however, prevents the rational de novo microalgae strain design. In this research, we describe the next-generation sequencing, de novo assembly, and functional annotation of the transcriptome of Ankistrodesmus sp. UCP0001. In total 48,867,830 high-quality sequence reads were de novo assembled into 38,414 unigenes (mean length = 508 bp, N50 = 1038 bp). Seventy-two percent of unigenes presented mapping information. Based on the KEGG pathway assignment, the fatty acids and the triacylglycerol biosynthesis pathways were reconstructed. Our results demonstrate that the synergy among high-throughput sequencing technologies and appropriate bioinformatics tools provides a fast, low-cost, and effective approach to generate invaluable functional genomic information in non-model microalgae species (e.g., Ankistrodesmus sp.). With the de novo assembled and annotated transcriptome we have successfully identified genes encoding enzymes and reconstructed metabolic pathways for the biosynthesis of fatty acids and triacylglycerols in this microalgae species. This genetic information could be used for the de novo microalgae strain design with desirable characteristics to produce biodiesel and capabilities for the biosynthesis of others valuable bioactive compounds of interest to the pharmacological, food, and cosmetic industries.