Obtención de plantas transgénicas de Digitalis purpurea L. (Plantaginaceae) que contienen el gen vep1 de Arabidopsis thaliana L.
Fecha
2018-05
Autores
Espinosa Antón, Adrian Alejandro
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Editor
Universidad Central "Marta Abreu" de Las Villas.Facultad de Ciencias Agropecuarias.Departamento de Biología
Resumen
Hasta la fecha, las plantas del género Digitalis son la principal fuente económicamente viable de cardenólidos. Estos metabolitos secundarios constituyen los medicamentos más extensamente empleados a nivel mundial en el tratamiento de la insuficiencia cardíaca y tienen interés farmacológico creciente, debido a sus potencialidades como anticancerígeno. La transformación genética es una herramienta promisoria para el desarrollo de nuevas estrategias biotecnológicas que propicien la sobreproducción de glucósidos cardiotónicos. Este estudio describe la transformación genética de discos foliares de Digitalis purpurea L., vía Agrobacterium tumefaciens, con el gen vep1 de Arabidopsis thaliana L. Para ello, se compararon los agentes selectivos higromicina B (15 mg L-1) y geneticina (70 mg L-1) durante el proceso de formación de callos, con la finalidad de elegir el antibiótico más efectivo en el proceso de selección. Posteriormente, se empleó la cepa de Agrobacterium C58C1RfR (pMP90), portadora del vector binario pAthspA2-vep1, para transferir el gen de interés a los segmentos foliares de D. purpurea. Los explantes seleccionados fueron transferidos a medio de cultivo de regeneración. La presencia de los transgenes p35S, hpt y vep1 en las plantas obtenidas fue comprobada mediante PCR. La selección con higromicina B durante el proceso de formación de callos de D. purpurea, es más eficiente que la realizada con geneticina. Además, se obtuvieron plantas transgénicas de D. purpurea que contenían el gen vep1 de A. thaliana. Este constituye el primer informe de la transformación genética de plantas del género Digitalis, con un gen involucrado en la biosíntesis de cardenólidos.
To date, plants of the genus Digitalis are the main economically viable source of cardenolides. These secondary metabolites are the most widely used drugs worldwide in the treatment of heart failure. Also cardenolides have increasing pharmacological interest due to their potential as anticancer agent. Genetic transformation is a promising tool for the development of new biotechnological strategies that propitiate the overproduction of cardiotonic glycosides. This study describes the genetic transformation of leaves segments of Digitalis purpurea L., via Agrobacterium tumefaciens, with Arabidopsis thaliana L. vep1 gene. First, the selective agents hygromycin B (15 mg L-1) and geneticin (70 mg L-1) were compared during the process of callus formation, in order to choose the most effective antibiotic in the selection process. Afterward, the strain of Agrobacterium C58C1RfR (pMP90), carrier of the binary vector pAthspA2-vep1, was used to transfer the gene of interest to the foliar segments of D. purpurea. The selected explants were transferred to regeneration medium. Successful transformation was confirmed by PCR analyses with p35S, hpt and vep1 genes specific primers. The selection with hygromycin B during the process of callus formation of D. purpurea is more effective. In addition, transgenic plants of D. purpurea containing vep1 gene were obtained. This is the first report of the genetic transformation of plants of the genus Digitalis, with a gene involved in the biosynthesis of cardenolides.
To date, plants of the genus Digitalis are the main economically viable source of cardenolides. These secondary metabolites are the most widely used drugs worldwide in the treatment of heart failure. Also cardenolides have increasing pharmacological interest due to their potential as anticancer agent. Genetic transformation is a promising tool for the development of new biotechnological strategies that propitiate the overproduction of cardiotonic glycosides. This study describes the genetic transformation of leaves segments of Digitalis purpurea L., via Agrobacterium tumefaciens, with Arabidopsis thaliana L. vep1 gene. First, the selective agents hygromycin B (15 mg L-1) and geneticin (70 mg L-1) were compared during the process of callus formation, in order to choose the most effective antibiotic in the selection process. Afterward, the strain of Agrobacterium C58C1RfR (pMP90), carrier of the binary vector pAthspA2-vep1, was used to transfer the gene of interest to the foliar segments of D. purpurea. The selected explants were transferred to regeneration medium. Successful transformation was confirmed by PCR analyses with p35S, hpt and vep1 genes specific primers. The selection with hygromycin B during the process of callus formation of D. purpurea is more effective. In addition, transgenic plants of D. purpurea containing vep1 gene were obtained. This is the first report of the genetic transformation of plants of the genus Digitalis, with a gene involved in the biosynthesis of cardenolides.
Descripción
Palabras clave
Cardenólidos, Genes, Agrobacterium Tumefaciens, Selection Marker Genes.