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Topicos Sobre Fisiologia, Toxicologia Y Biologia TOPICOS SOBRE FISIOLOGIA, TOXICOLOGIA Y BIOLOGIA MOLECULAR 831 AISLAMIENTO Y CARACTERIZACIÓN MOLECULAR DE GENES QUE CODIFICAN PARA QUITINASAS Y PROTEASAS DE HONGOS ENTOMOPATÓGENOS Isolation and molecular characterization of chitinases and proteases encoding genes from entomopathogenic fungi Miguel Silva-Flores1. Sergio Casas-Flores1. Ovidio Díaz-Gómez2, y Clara Monreal- Vargas2. 1División de Biología Molecular. Instituto Potosino de Investigación Científica y Tecnológica (IPICyT). Camino a la Presa San José No. 2055. Col. Lomas 4a. Sección, C.P. 7821. San Luis Potosí, SLP. Tel: + (444) 834 2000 Ext. 2079. Fax + (444) 834 2010 E-mail: miguel.silva @ipicyt.edu.mx; [email protected]; 2Fac. de Agronomía de la U.A.S.L.P., A. Obregón 64, Centro, 78000, San Luis Potosí, S.L.P. [email protected] Palabras Clave: Entomopatógenos, quitinasa, proteasa. Introducción En México se considera a Bactericera cockerelli (Sulc) como vector del agente causal de la fitoplasmosis en solanáceas (Garzón, 2002), mientras que en el resto del mundo es importante por el efecto que causan las toxinas que inyecta al alimentarse. En muchas ocasiones, en complejo con mosca blanca, esta especie devasta amplias zonas de cultivo, principalmente solanáceas, por lo que para su control se usa infinidad de elementos que van desde insecticidas químicos hasta productos biológicos. El Manejo Integrado de Plagas (MIP) contempla, además de insecticidas organosintéticos, la liberación de enemigos naturales, la aplicación de productos bioracionales y el uso de organismos entomopatógenos. Existen parasitoides y depredadores que han demostrado ser eficientes en el control de insectos plaga, además de productos bioracionales como los ácidos grasos, extractos vegetales, repelentes, reguladores de crecimiento, etc. Adicionalmente, se han evaluado hongos entomopatógenos, como Entomophtora virulenta, Beauveria bassiana. Verticillium lecanii, Metarhizium anisopliae y Paecilomyces fumosoroseus. Ese último reviste particular importancia dado que infecta a la mosquita blanca con excelentes resultados, en algunos casos, mejores a los obtenidos con control químico. En la actualidad, las herramientas que proporciona la biología molecular han permitido la manipulación genética de algunas cepas de hongos para potenciar la efectividad que estos presentan de manera natural. P. fumosoroseus ha demostrado ser efectivo para el control de la mosquita blanca, destacando por las epizootias que causa, en muchas ocasiones, de manera natural. La forma infectiva de los hongos son las conidias y las blastoconidias, que pueden llegar a penetrar al insecto a través de la cavidad bucal, de los espiráculos o de la cutícula (James, 2001). Cuando el hongo penetra a través de la cutícula, el mecanismo infectivo incluye la adhesión del conidio a la cutícula donde germina y forma una estructura llamada apresorio, el cual hace presión mecánica, junto con la secreción de hidrolasas; principalmente, proteasas, lipasas y quitinasas, que rompen la cutícula, permitiendo la entrada del hongo al hemocele del insecto. Este hongo posee la capacidad de evadir la respuesta de defensa del insecto y de digerir los órganos internos hasta causarles la muerte (Khachatourians, 1996; citado por Sánchez, 2004). Por lo anterior, es importante identificar y caracterizar los genes que 832 codifiquen estas enzimas para entender los mecanismos de patogenicidad, y posteriormente, mediante ingeniería genética, modificarlos para incrementar su virulencia hacia el huésped, como ya se ha hecho con algunos entomopatógenos (Metarhizium anisopliae. y Beauveria bassiana). Sin embargo, es importante contar con un amplio rango de genes caracterizados, para emplearlos de acuerdo a las necesidades, es decir, se requiere generar un banco que permita desarrollar herramientas mas diversas y competitivas para el control biológico de insectos. Por lo anterior se planteó el siguiente objetivo: Clonar y caracterizar genes que codifican para quitinasas y proteasas de hongos entomopatógenos. Materiales y Método Se diseñaron oligonucleótidos degenerados en regiones consenso de quitinasas y proteasas de hongos entomopatógenos, con la finalidad de amplificar por PCR las regiones conservadas en el genoma de los hongos. Adicionalmente, se diseñaron oligonucleótidos sobre la secuencia obtenida, para amplificar los genes completos de proteasas y quitinasas de P. fumosoroseus, mediante la técnica de DNA-walking. Los productos amplificados por PCR fueron clonados en pGEM-Teasy y se seleccionaron algunas clonas que fueron secuenciadas por el método de Sanger. Las secuencias se analizaron utilizando el algorítmo BLASTX de NCBI. Mediante microscopia se identificó un hongo perteneciente al genero Cladosporium sp., el cual fue identificado parasitando al psilido del eucalipto Glycaspis brimblecombei MOORE. Con la finalidad de determinar la identidad de este hongo, se procedió a realizar la amplificación por PCR de los ITS y posteriormente se transformó y se clono en pGEM- Teasy. Se seleccionaron algunas clonas, para su posterior secuenciación. Resultados y Discusión Por medio de la Reacción en Cadena de la Polimerasa (PCR), utilizando oligonucleótidos degenerados diseñados sobre regiones conservadas para quitinasas y proteasas de diferentes organismos, se obtuvieron las secuencias parciales de dos genes de P. fumosoroseus; uno de ellos codifica para una quitinasa, mientras que el segundo gen codifica para una probable proteasa. Posteriormente, utilizando el método de DNA Walking, se amplificó un fragmento de 1672 bp, que corresponde al gen completo de una posible quitinasa, lo cual se determinó utilizando el algorímo GENSCAN. Con el análisis tipo BLASTX se encontró que la secuencia de P. fumosoroseus presenta una identidad de 85% respecto a una quitinasa codificada por el entomopatógeno Lecanicillium lecanii (Fig. 1). Dado que ha la fecha no se había caracterizado el gen que codifica para la quitinasa en P. fumosoroseus, este es el primer reporte que se hace de esta naturaleza. Lo anterior es de importancia en la investigación, puesto que una vez caracterizado el gen, puede manipularse para generar cepas más virulentas. Analizando la secuencia amplificada por PCR con oiligonucleótidos universales para las regiones intergénicas de los RNA ribosomales de hongos, con respecto a la cepa de Cladosporium, se encontró mediante BLASTN, en el Gene bank, que la secuencia aislada alineó con el gen 18S ribosomal que pertenece el genero Cladosporium cladosporioides. Este hongo potencialmente puede ser un agente de control biológico, ya que, inicialmente se aisló de G. brimblecombei en eucalipto (Fig. 2); también se ha citado parasitando pupas de Cameraria ohridella (Samek et al., 2006). 833 Figura. 1. Alineamiento mediante BLASTN de la secuencia del DNA geonómico de P. fumosoroseus a b Fig. 2. Ninfas de Bactericera cockerelli parasitadas por el hongo Cladosporium cladosporioides aislado del psilido del eucalipto. a) fase inicial de la infección b) ninfa 8 DDA . Las perspectivas de la investigación son: Cuantificar la expresión del gen de la quitinasa en diferentes condiciones de cultivo por medio de la técnica de PCR en tiempo real. Clonar genes para quitinasas y proteasas en C. cladosporioides. Transformar P. fumosoroseus con Agrobacterium tumefaciens, para interrumpir y sobreexpresar los genes de quitinasas y proteasas para determinar su papel durante el proceso de infección. Evaluar in vitro la actividad insecticida de las cepas, transformantes generadas de ambos hongos. Evaluar in vivo, bajo condiciones controladas, la efectividad biológica de las cepas transformantes de P. fumosoroseus sobre mosca blanca, y sobre B. cockerelli las de C. cladosporioides. Conclusiones Se logró la secuenciación de un gen de P. fumosoroseus que codifica para una quitinasa, similar en 85% a una codificada por L. lecanii. La secuencia aislada de Cladosporium es parte del 18S ribosomal, que pertenece el genero C. cladosporioides. Literatura Citada Garzón, T. J. A. 2002. El “Pulgón saltador “o la Paratrioza una amenaza para la horticultura de Sinaloa. Memoria de taller sobre Paratrioza cockerelli Sulc. como plaga y vector de fitoplasmas en hortalizas. Culiacán Sinaloa. Mexico, 100 pp. 834 James, R. R. 2001. Effects of exogenous nutrients on conidial germination and virulence against the silver leaf whitefly for two hyphomycetes. J. invertebr Pathol. 77 pp 99- 107. Pucheta, D. M. Flores M. A., Rodríguez N. S. y De la Torre M. 2006. Mecanismo de acción de los hongos entomopatógenos. INCI., vol. 31:12. pp. 856-860. Sánchez, M. R. 2004. Estudios sobre la reproducción asexual del hongo entomopatógeno Paecilomyces fumosoroseus. Tesis para obtener el grado de Doctor en Biotecnología. Centro de Investigación y de estudios Avanzados del Instituto Politécnico Nacional. Unidad Zacatenco. México, D.F. T. Samek, D. Novotný y L. Jankovský. 2006. Infection of wintering pupae of horse- chestnut leaf miner. Cameraria ohridella Deschka et Dimić. by Verticillium lecanii (Zimmerman) Viégas. J. Forest Science, 52(3): pp. 136–140 835 DIEZ AÑOS DE MONITOREO DE LA RESISTENCIA EN POBLACIONES MEXICANAS DE Helicoverpa zea BODDIE A LA TOXINA Cry1Ac QUE PRODUCE EL ALGODONERO TRANSGÉNICO BOLLGARD® I Ten years of monitoring the resistance in Mexican Populations of Helicoverpa zea Boddie to Cry1Ac toxin that produces the transgenic cotton Bollgard® I Sotero Aguilar-Medel1 y J. Concepción Rodríguez-Maciel2. 1Universidad Autónoma del Estado de México. Centro Universitario Tenancingo. Tenancingo, Edo. de México. [email protected].
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