Revista latinoamericana de química
versão impressa ISSN 0370-5943
MARTINEZ-HERNANDEZ, Aída; MENA-ESPINO, Ma. Esther; HERRERA-ESTRELLA, Alfredo H. e MARTINEZ-HERNANDEZ, Pedro. Construcción de bibliotecas de ADNc y análisis de expresión génica por RT-PCR en agaves. Rev. latinoam. quím [online]. 2010, vol.38, n.1, pp. 21-44. ISSN 0370-5943.
Mexico is the center of origin and diversification of agave, a highly valued crop in the agroindustry, as well as in Mexican culture. Agaves present unique biological characteristics, such as their high tolerance to abiotic stress that allows them to grow under extreme environmental conditions, and their secondary metabolism that leads to the production of molecules of biotechnological interest. However, the necessary tools to carry out molecular physiology studies in Agave, and disclose the subjacent molecular basis of their particular metabolic capacity, have not been developed. Here we evaluate the performance of a series of commercially available methods for RNA extraction and mRNA purification that may be suitable for the construction of cDNA libraries derived from different A. tequilana tissues, a process described in detail. In addition, we report sequences and methods for the analysis of gene expression using RT-PCR for physiologically relevant agave genes. Expression of rbcS, encoding the small subunit of the RuBisCO; a gene encoding an NADP+ dependent malic enzyme; a gene encoding 1-SST fructosyltransferase; and a gene coding for a LEA-like abiotic stress related protein of A. tequilana, was analyzed using this methods. The methods set forth herein, and the libraries generated will subsequently be applied to molecular physiology, functional genomics, and transcriptome analysis of agaves.
Palavras-chave : Agave tequilana; RNA extraction; cDNA libraries; gene expression; RT-PCR.