“La Uva” (Sinaloa) Con Vegetación De Selva

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“La Uva” (Sinaloa) Con Vegetación De Selva INSTITUTO POLITÉCNICO NACIONAL CENTRO INTERDISCIPLINARIO DE INVESTIGACIÓN PARA EL DESARROLLO INTEGRAL REGIONAL UNIDAD SINALOA Biodiversidad de la microbiota rizosférica de dos especies solanáceas: Solanum lycopersicon, L. y Datura spp. (tomate y toloache) TESIS QUE PARA OBTENER EL GRADO DE MAESTRÍA EN RECURSOS NATURALES Y MEDIO AMBIENTE PRESENTA RAQUEL LÓPEZ RIVERA GUASAVE, SINALOA, MÉXICO. DICIEMBRE DE 2011. Agradecimientos a proyectos El trabajo de tesis se desarrolló en el lanoratoriao de Ecología Molecular de la Rizosfera en el Departamento de Biotecnología Agrícola del Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (CIIDIR) Unidad Sinaloa del Instituto Politécnico Nacional (IPN) bajo la dirección del Dr. Ignacio Eduardo Maldonado Mendoza, E-mail: [email protected], domicilio laboral: Boulevard Juan de Dios Bátiz Paredes, No. 250 CP: 81100, Colonia: San Joachin, Ciudad: Guasave, Sinaloa, Fax: 01(687)8729625, Teléfono: 01(687)8729626. El presente trabajo fue apoyado económicamente a través del CONABIO (Con número de registro GE019). El alumno/a Raquel López Rivera fue apoyado con una beca CONACYT con clave 332252. AGRADECIMIENTOS A Dios por estar siempre a mi lado y darme la fuerza necesaria para seguir adelante y llegar a este momento de mi vida. A mis padres Roberto y Tomasa por darme la vida y enseñarme a vivirla, por todo el apoyo que me han brindado y por los consejos que me han guiado por el camino correcto. A mis hermanos Miriam, Miguel y Roberto que siempre han estado conmigo apoyándome de una u otra manera y en especial a mi hermana Carolina†, a pesar del tiempo te sigo extrañando. A mi director de tesis el Dr. Ignacio E. Maldonado Mendoza por permitirme formar parte de su grupo de trabajo y por toda su enseñanza, apoyo y regaños a lo largo de mi maestría que me han ayudado a superarme de manera profesional y personal. A mi comité tutorial por sus consejos y recomendaciones para que este trabajo se llevara a cabo de la mejor manera. Al CIIDIR Sinaloa por abrirme sus puertas y permitirme ser parte de este centro que me ha permitido crecer profesionalmente y en donde he conocido a grandes amigos. A Manuel por llegar a mi vida, por ser ese rayito de sol que ilumina mis días y que siempre me anima a seguir adelante, gracias por todo tu apoyo, consejos y momentos de alegría. A mis amigos Lucy, Edalhí, Paola, Judith, Andrés, Daniel, Glenda, Karla, Alicia y Alejandra por acompañarme a lo largo de esta aventura, por todos los momentos divertidos que pasamos juntos, por los momentos de risa y los de lagrimas, por hacer que los momentos de estrés fueran más leves y por las horas de risoterapia a la hora de de la comida, gracias porque con ustedes este recorrido fue más leve. Al Dr. Carlos Ligne por su apoyo y asesorías en el análisis de mis secuencias. A Sindy Galaviz por su apoyo en la incorporación de los datos en la base de datos. A mis profesores por todas sus enseñanzas y por compartirnos siempre sus experiencias. A mis compañeros de generación, por ser parte de esta recorrido y por los pocos o muchos momentos que compartimos a lo largo de la maestría. ÍNDICE GLOSARIO…………………………………………………………………………….....IV ÍNDICE DE FIGURAS.............................................................................................VI ÍNDICE DE CUADROS...........................................................................................IX RESUMEN………………………………………………………………………………..X ABSTRACT............................................................................................................XII 1. INTRODUCCIÓN………………………………………………………………….......1 2. ANTECEDENTES…………………………….…………………………..………......3 2.1. El suelo…………………………………………………………………........3 2.2. La rizosfera……………………………………………………………..........3 2.3. Los microorganismos del suelo……………………………………..….....6 2.4. Ecosistema, hábitat y formas de organización de los microorganismos....................................................................................7 2.5. Técnicas moleculares para el análisis de la diversidad de los microorganismos del suelo…………..…………………………………….9 2.6. Estudio de las comunidades de la microflora del suelo………..........12 2.7. Estudio de la microflora de la rizosfera de tomate en un área de cultivo...................................................................................................13 2.8. Estudios sobre microorganismos del suelo en “La Uva”………….......16 2.9. Datura stramonium, L. y otras solanáceas de importancia en Sinaloa.................................................................................................17 2.10. Zona de preservación ecológica del centro de población “La Uva...20 3. JUSTIFICACIÓN………………………………………………………………...…...24 4. OBJETIVOS……………………………………………………………….................25 4.1. Objetivo general …...…………………………………….………............25 4.2. Objetivos específicos…...………...………………………...…….............25 5. HIPÓTESIS……………………………………..………………………………....….26 6. MATERIALES Y MÉTODOS……………………..………………………...............27 6.1. Esquema general de la metodología………………….............……......27 6.2. Muestreo del suelo......…………………......……………………….........27 I 6.3. Creación del banco de microorganismos aislados de la rizosfera de Datura stramonium, L………………….................................................30 6.4. Identificación molecular de los especímenes del banco de germoplasma de tomate y toloache……………….......................…....31 6.4.1 Extracción de ADN genómico...................................................31 6.4.2. Reacción en cadena de la polimerasa….................................32 6.4.3. Visualización de los productos de PCR………………….........33 6.4.4. Purificación de los productos de PCR, cuantificación y secuenciación del ADN…..................………………………….34 6.4.5. Análisis de las secuencias…………………….………..............34 6.5. Obtención del banco de clonas de ADN ribosomal que incluye organismos cultivables y no cultivables………………………..........….34 6.5.1. Extracción del ADN genómico de suelo…………………........34 6.5.2. Reacción en cadena de las polimerasa……………….…........35 6.5.3. Electroforesis del producto de PCR……………….……...........37 6.5.4. Precipitación del producto de PCR……………......….…….….37 6.5.5. Clonación de los productos de PCR………………………......38 6.5.6. Transformación de los productos de PCR…………................38 6.5.7. Purificación del ADN plasmídico………....…………................39 6.5.8. Digestión del ADN plasmídico…………….…...……................39 6.5.9. Secuenciación y análisis de las secuencias…………….........40 6.6. Análisis de los datos obtenidos y comparación de la microflora de la rizosfera de Datura stramonium, L. y de la rizosfera de tomate……...41 6.7. Incorporación de la información obtenida en una base de datos........42 7. RESULTADOS ……………...………………………………….....………...............43 7.1. Análisis físico-químico del suelo...........................................................43 7.2. Creación del banco de microorganismos ………...………………….....44 7.3. Identificación molecular de los especímenes del banco de tomate y toloache……………………………………………………………………44 7.3.1. Banco de tomate……………………………………...................44 7.3.2. Banco de toloache….…………………………………................47 II 7.4. Obtención del banco de clonas de ADNr de la rizosfera de Datura stramonium, L………………………………………………………………51 7.4.1. Análisis de la microbiota del banco de clonas de origen procariota de la rizosfera de toloache……………………….....54 7.4.2. Análisis de la microbiota del banco de clonas de origen procariota de la rizosfera de toloache………………………….61 7.4.3. Análisis comparativo de la diversidad de la rizosfera de Toloache y Tomate……………….………………………………69 7.5. Incorporación de los datos de la rizosfera de tomate y toloache en la base de datos…….……………………………………………………….....79 8. DISCUSIÓN………………………………………………………………….……….82 8.1. Obtención del banco de microorganismos y del banco de clonas de microorganismos asociados a la rizosfera de toloache……….…........82 8.2. Identificación molecular de los especímenes del banco de germoplasma de tomate……………………………………………….....83 8.3. Análisis de diversidad de organismos cultivables y no cultivables empleando bibliotecas de clonas procariotas y eucariotas de la rizosfera de toloache...........................................................................86 8.3.1 Análisis de la biblioteca de clonas procariotas……..……….....86 8.3.2 Análisis de la biblioteca de clonas eucariotas……......…..........88 8.4. Análisis comparativo de la diversidad de la rizosfera de toloache y tomate..................................................................................................90 8.5. Incorporación de los datos de la rizosfera de tomate y toloache en la base de datos……….……………………………………………………...96 9. CONCLUSIONES………………………………………………….……..…............97 10. BIBLIOGRAFÍA…………………………………………………………...………...99 11. ANEXOS…………………………………………………………...………...........115 III GLOSARIO Abundancia de especies de una comunidad. Es el número de individuos de las especies que conforman a la comunidad. Una alta abundancia relativa de un grupo en una comunidad microbiana puede indicar que el grupo es numéricamente dominante o que está creciendo rápidamente dentro de esa comunidad. Acuminado. Se dice de un órgano terminado en punta (hojas, frutos). Alcaloides derivados del tropano. Son metabolitos secundarios que contienen en la estructura de sus moléculas, átomos de nitrógeno secundario, terciario y cuaternario. Algunos de estos alcaloides funcionan como fitoalexinas ó en la interacción planta-insecto, algunos han sido utilizados en la industria farmacéutica. Biodiversidad
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