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CARACTERIZACIÓN Y ANÁLISIS DE EXPRESIÓN DE LOS GENES NBS-LRR EN Agave Tequilana

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CARACTERIZACIÓN Y ANÁLISIS DE EXPRESIÓN DE LOS GENES NBS-LRR EN Agave Tequilana Centro de Investigación Científica de Yucatán, A.C. Posgrado en Ciencias Biológicas CARACTERIZACIÓN Y ANÁLISIS DE EXPRESIÓN DE LOS GENES NBS-LRR EN Agave tequilana Tesis que presenta GERARDO CAMPOS RIVERO En opción al título de DOCTOR EN CIENCIAS (Ciencias Biológicas: Opción Biotecnología) Mérida, Yucatán, México 2020 DECLARACIÓN DE PROPIEDAD Declaro que la información contenida en la sección de Materiales y Métodos Experimentales, los Resultados y Discusión de este documento proviene de las actividades de experimentación realizadas durante el período que se me asignó para desarrollar mi trabajo de tesis, en las Unidades y Laboratorios del Centro de Investigación Científica de Yucatán, A.C., y que a razón de lo anterior y en contraprestación de los servicios educativos o de apoyo que me fueron brindados, dicha información, en términos de la Ley Federal del Derecho de Autor y la Ley de la Propiedad Industrial, le pertenece patrimonialmente a dicho Centro de Investigación. Por otra parte, en virtud de lo ya manifestado, reconozco que de igual manera los productos intelectuales o desarrollos tecnológicos que deriven o pudieran derivar de lo correspondiente a dicha información, le pertenecen patrimonialmente al Centro de Investigación Científica de Yucatán, A.C., y en el mismo tenor, reconozco que si derivaren de este trabajo productos intelectuales o desarrollos tecnológicos, en lo especial, estos se regirán en todo caso por lo dispuesto por la Ley Federal del Derecho de Autor y la Ley de la Propiedad Industrial, en el tenor de lo expuesto en la presente Declaración. ________________________________ GERARDO CAMPOS RIVERO Este trabajo se llevó a cabo en la Unidad de Biotecnología del Centro de Investigación Científica de Yucatán, bajo la dirección del Dr. Lorenzo Felipe Sánchez Teyer. AGRADECIMIENTOS Al Centro de Investigación Científica de Yucatán, en especial a la unidad de biotecnología, por proporcionar los equipos necesarios para el desarrollo de este trabajo. Al CONACYT, por la beca otorgada no. 2888867. Centro de Biotecnología y Genómica del instituto politécnico nacional, por el espacio y el soporte técnico brindado durante la estancia para la evaluación de la patogenicidad de hongos asociados A. tequilana Al laboratorio del Dr. Felipe A. Vázquez flota y a la M en C. Miriam Monforte González, por el espacio y soporte técnico brindado en el laboratorio durante el análisis de expresión de genes A la Dra. Claudia patricia Larralde corona, por su apoyo y asesoría durante la estancia en el IPN. A la Dra. Yahaira Tamayo Ordoñez, por la capacitación técnica del equipo Illumina eco real-time. A mi asesor Dr. Felipe Sánchez Teyer, por la información académica brindada A mi coasesor Dr. José A. Narváez Zapata, por su importante colaboración en el desarrollo del trabajo. A la técnica Escalante Erosa, por el apoyo técnico durante el doctorado. A las técnicas Erika a. De la cruz Arguijo y Socorro Ramírez, por el soporte técnico durante la estandarización de las técnicas enzimáticas, así como para la conservación de las cepas de hongos. A mi comité evaluador y sinodales, Dra. Montserrat Calderón Santoyo, Dra. Cecilia Hernández Zepeda, Dra. Blondy Canto Canché, Dr. Luis Alfonso Sáenz, Dr. Manuel Martínez Estévez, Dra, Renata Rivera Madrid, Dr. José a. Narváez zapata y al Dr. Lorenzo Felipe Sánchez Teyer, por la asesoría brindada. DEDICATORIAS Agradezco a Dios, por la oportunidad de concluir uno más de mis objetivos y por haberme guiado en el camino. A mis padres Ofelia Ma. Rivero y Efraín Campos, quienes con su ejemplo, amor, dedicación y apoyo incondicional ayudaron a concluir este proyecto. A mi novia Samantha, por su apoyo, compresión y ayuda durante todo el proceso. Agradezco a mis hermanos Efraín y Gibran quienes estuvieron junto a mí durante todo el proceso apoyando en todo momento. A mis compañeros de laboratorio, por todo el apoyo brindado durante el proceso. ÍNDICE ÍNDICE LISTADO DE CUADROS ................................................................................................ vii LISTADO DE FIGURAS ................................................................................................. viii ABREVIATURAS ............................................................................................................. xi RESUMEN ......................................................................................................................... 1 ABSTRACT ....................................................................................................................... 2 INTRODUCCIÓN ............................................................................................................... 1 CAPÍTULO I ...................................................................................................................... 3 ANTECEDENTES ............................................................................................................. 3 1.1 Generalidades del género Agave L. .......................................................................... 3 1.2 Problemática: El Agave frente al estrés biótico ....................................................... 5 1.3 Otros microorganismos presentes en A. tequilana y su potencial patogénico .... 6 1.4 Mecanismos de defensa ............................................................................................ 7 1.4.1 Respuesta inducible ............................................................................................... 7 1.4.2 Respuesta sistémica adquirida (SAR) ................................................................... 9 1.4.3 Genes R (NBS-LRR) ................................................................................................ 9 1.4 Relación infección-expresión de genes NBS-LRR ................................................ 12 1.5 Genes NBS-LRR y mejoramiento vegetal ............................................................... 15 JUSTIFICACIÓN ............................................................................................................. 16 HIPÓTESIS ..................................................................................................................... 16 i ÍNDICE OBJETIVO GENERAL .................................................................................................... 17 OBJETIVOS ESPECÍFICOS ........................................................................................... 17 ESTRATEGIA EXPERIMENTAL ..................................................................................... 18 CAPÍTULO II ................................................................................................................... 19 IDENTIFICACIÓN DE PATÓGENOS POTENCIALES ASOCIADOS A Agave tequilana var. azul* ......................................................................................................................... 19 2.1 INTRODUCCIÓN ....................................................................................................... 19 2.2 MATERIALES Y MÉTODOS ..................................................................................... 22 2.2.1 Aislamiento de hongos ......................................................................................... 22 2.2.2 Extracción de DNA, PCR y secuenciación .......................................................... 22 2.2.3 Clonación .............................................................................................................. 24 2.2.3.1 Secuenciación y análisis bioinformático de las regiones ITS y 26s ............... 24 2.2.3.2 Análisis filogenéticos ........................................................................................ 25 2.2.3.3 Conservación y selección de los microorganismos ....................................... 26 2.2.4 Ensayo de patogénesis in vitro............................................................................ 26 2.2.5 Actividad enzimática de los extractos crudos. ................................................... 27 2.2.5.1 Obtención del extracto enzimático. .................................................................. 28 2.2.5.1.1 Contenido de proteínas por el método de Bradford. .................................... 28 2.2.5.1.2 Contenido de azúcares en el extracto crudo. ............................................... 28 2.2.5.2 Evaluación de la actividad pectinasa. .............................................................. 28 2.3 RESULTADOS .......................................................................................................... 29 ii ÍNDICE 2.3.1 Aislamiento e identificación de organismos potencialmente patógenos asociados a Agave tequilana var. azul ......................................................................... 29 2.3.2 Identificación molecular de los organismos purificados ................................... 31 2.3.3 Análisis filogenético de las regiones D1- D2 (LSU) y IST1-5.8s-ITS2 del rDNA 32 2.3.4 Ensayo de patogenicidad ..................................................................................... 40 2.4 DISCUSIÓN ............................................................................................................... 44 2.4.1 Identificación de los hongos aislados ................................................................. 44 2.4.2 Ensayo de patogenicidad ..................................................................................... 48 CAPÍTULO III .................................................................................................................. 53 ANÁLISIS
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