Metodologías para el análisis de expresión y cuantificación de proteínas conferidas en tejidos de cultivos GM

EnglishThe Cry toxins of Bacillus thuringiensis(Bt) are used as bioinsecticides in agriculture. Reliable methods are needed to analyze the characteristics that are conferred to genetically modified (GM) plants during their development, before commercialization. The objective of the work was to standardize methodologies for the analysis of the expression of genes and proteins conferred to GM cotton during its different phenological stages in the field. As the main practical application of the study, the standardized methodologies can be used in the characterization of GM crops that are developed and for which it is necessary to carry out their risk analysis. To do this, samples of plant tissue were processed in different phenological stages obtained from GM cotton commercial farms in the Yaqui Valley. In the different tissues, gene and protein expression was quantified by RTq-PCR and Elisa analysis, respectively. The results obtained show variation in the expression of the genes conferred throughout the development of the same variety and between the different sites where the crops were located. The highest levels of expression were identified, as expected, in the early stages of the culture (average values of 8.5 μg g-1for Cry1Ac and 63.1 μg g-1for Cry2Ab) compared with that observed in late or mature stages (mean values 0.05 and 0.3 μg g-1for Cry1Ac and Cry2Ab, respectively). Therefore, it is concluded that the RTq-PCR and ELISA techniques are suitable to evaluate the spatial and temporal expression of genes thatare conferred to GM plants, information required to characterize the exposure to the hazard and to perform the risk analysis. MultipleLas toxinasCrydeBacillus thuringiensis(Bt) sonutilizadas como bioinsecticidas en la agricultura.Se precisa de metodos confiables que permitan analizar las caracteristicas que se confieren a las plantas geneticamente modificadas (GM)durante su desarrollo, antes de su comercializacion.El objetivo del trabajo fue estandarizar metodologias para el analisis de la expresion de genes y proteinas conferidas al algodonero GM durante sus diferentes etapas fenologicas en campo. Como principal aplicacion practica del estudio realizado, las metodologias estandarizadas sepodran emplear en la caracterizacion de cultivos GM que se desarrollen y para los cuales sea necesario realizar su analisis de riesgo. Para ello se procesaron muestras de tejido vegetalen diferentes etapas fenologicasobtenidas de predios comerciales de algodoneroGM en elValle del Yaqui.En los diferentes tejidosse cuantifico la expresion genicay proteica mediante analisisRTq-PCR y Elisa, respectivamente. Los resultados obtenidos muestranvariacion enla expresion de los genes conferidos a lo largo del desarrollode la misma variedadyentrelos diferentes sitios donde se ubicaron los cultivos. Los mayores niveles deexpresion se identificaron,como se esperaba,en las etapas tempranasdel cultivo(valores medios de 8.5 μg g-1para Cry1Ac y 63.1 μg g-1para Cry2Ab) comparados con loobservado en etapas tardias o maduras (valores medios de 0.05 y 0.3 μg g-1para Cry1Ac y Cry2Ab, respectivamente). Por lo tanto,se concluye que las tecnicas de RTq-PCR y ELISA son adecuadas para evaluar la expresion espacial y temporal de genes que se confieran a plantas GM, informacion requerida para caracterizar laexposicional peligro y poder realizar el analisis de riesgo.