Abstract
Together with their undeniable role in the ecology of arid and semiarid ecosystems, Agave species are emerging as a model to dissect the relationships between crassulacean acid metabolism and high efficiency of light and water use, and as an energy crop for bioethanol production. Transcriptome resources from economically valuable Agaves species, such as Agave tequilana and A. salmiana, as well as hybrids for fibers, are now available, and multiple gene expression landscape analyses have been reported. Key components in molecular mechanisms underlying drought tolerance could be uncovered by analyzing gene expression patterns of roots. This study describes an efficient protocol for high-quality total RNA isolation from phenolic compounds-rich Agave roots. Our methodology involves suitable root handling and collecting in the field and using saving-time commercial kits available. RNA isolated from roots free of lignified out-layers and clean cortex showed high values of quality and integrity according to electrophoresis and microfluidics-based platform. Synthesis of long full-length cDNAs and PCR amplification tested the suitability for downstream applications of extracted RNA. The protocol was applied successfully to A. tequilana roots but can be used for other Agave species that also develop lignified epidermis/exodermis in roots.
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Acknowledgements
This work was funded by CONACyT (Grant 1049) and Colpos. Luis F. Maceda-Lopez is grateful for a M.Sc. fellowship from CONACyT. Eleazar Garcia-Hernández and Dalia C. Moran Velazquez were supported by an undergraduate scholarship from CONACyT (Grant 1049). Authors especially grateful to Victor H. Fernandez-Carrillo, manager of Santa Genoveva farm, for supporting during Agave samples collecting.
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LFML, JLVA, DCMV, and FAC designed and performed the RNA extraction, quantification, and quality RNA analysis. JLVA, EGH, LRL, DHD, and MACV performed RT-PCR analysis. Lignin deposition and SEM analysis were carried out by LFML, SBAC, LRL, and FAC. Data mining was performed by LFML, EAD, JS, LT, ILR, EGC, and FAC. LFML, LT, EGC, ILR, JS, and FAC discussed results. LFML and FAC conceived and designed the research and wrote the paper. All authors read and approved the final manuscript.
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Maceda-López, L.F., Villalpando-Aguilar, J.L., García-Hernández, E. et al. Improved method for isolation of high-quality total RNA from Agave tequilana Weber roots. 3 Biotech 11, 75 (2021). https://doi.org/10.1007/s13205-020-02620-8
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DOI: https://doi.org/10.1007/s13205-020-02620-8