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CARACTERIZACIÓN MOLECULAR DE CEPAS NATIVAS COLOMBIANAS DE Azotobacter Spp

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CARACTERIZACIÓN MOLECULAR DE CEPAS NATIVAS COLOMBIANAS DE Azotobacter Spp CARACTERIZACIÓN MOLECULAR DE CEPAS NATIVAS COLOMBIANAS DE Azotobacter spp. MEDIANTE EL ANALISIS DE RESTRICCION DEL DNA RIBOSOMAL 16S DIEGO JAVIER JIMÉNEZ AVELLA PONTIFICIA UNIVERSIDAD JAVERIANA FACULTAD DE CIENCIAS BÁSICAS MICROBIOLOGÍA INDUSTRIAL Bogotá, D. C. Colombia AGOSTO DE 2007. CARACTERIZACIÓN MOLECULAR DE CEPAS NATIVAS COLOMBIANAS DE Azotobacter spp. MEDIANTE EL ANALISIS DE RESTRICCION DEL DNA RIBOSOMAL 16S DIEGO JAVIER JIMÉNEZ AVELLA TRABAJO DE GRADO PRESENTADO COMO REQUISITO PARCIAL PARA OBTAR EL TITULO DE MICROBIOLOGO INDUSTRIAL DIRECTOR: JOSE SALVADOR MONTAÑA CO-DIRECTORA: MARIA MERCEDES MARTINEZ PONTIFICIA UNIVERSIDAD JAVERIANA FACULTAD DE CIENCIAS BÁSICAS MICROBIOLOGÍA INDUSTRIAL Bogotá, D. C. Colombia AGOSTO DE 2007 NOTA DE ADVERTENCIA Articulo 23 de la resolución No 13 de julio de 1946 “La universidad no se hace responsable por los conceptos omitidos por sus alumnos en sus trabajos de tesis. Solo se velara porque no se publique nada contrario al dogma y a la moral católica y porque las tesis no contengan ataques personales contra persona alguna, antes bien se vea en ellas el anhelo en buscar la verdad y la justicia” CARACTERIZACIÓN MOLECULAR DE CEPAS NATIVAS COLOMBIANAS DE Azotobacter spp. MEDIANTE EL ANALISIS DE RESTRICCION DEL DNA RIBOSOMAL 16S DIEGO JAVIER JIMÉNEZ AVELLA APROBADO José Salvador Montaña Msc. María Mercedes Martínez Msc. Director Codirectora María del Pilar Márquez Msc. Jurado 1 Andrea Juliana Mantilla Microbióloga Industrial Jurado 2 Bogotá. D. C. Colombia AGOSTO DE 2007. CARACTERIZACIÓN MOLECULAR DE CEPAS NATIVAS COLOMBIANAS DE Azotobacter spp. MEDIANTE EL ANALISIS DE RESTRICCION DEL DNA RIBOSOMAL 16S DIEGO JAVIER JIMÉNEZ AVELLA APROBADO Ángela Umaña MPhil. David Gómez Msc. Decana Académico Director de Carrera Bogotá. D. C. Colombia AGOSTO DE 2007 DEDICATORIA A mis padres por todo su amor, su esfuerzo constante durante mi carrera, su comprensión, su ayuda y apoyo incondicional y único, y por creer en mí cada momento en esta gran etapa de mi vida. AGRADECIMIENTOS A mis directores José Salvador Montaña y María Mercedes Martínez, por su apoyo incondicional, por compartir sus invaluables enseñanzas y conocimientos conmigo y por acompañarme en este valioso paso en mi vida. A todas las personas del Laboratorio de Microbiología Ambiental y Suelos de la Pontificia Universidad Javeriana, por su colaboración y por haber permitido el desarrollo de esta investigación. A los investigadores de las Unidades de Saneamiento y Biotecnología Ambiental (USBA) y Biotecnología Vegetal (UBV) por su colaboración y asesoría. Al Ingeniero agrónomo Andrés Siabato por su colaboración y asesoría en el muestreo. A mis hermanos Julis, Caro y Chepe, por su apoyo y cariño en toda mi carrera. A mi linis por amarme tanto, por su compañía, por su comprensión y por ser un apoyo incondicional. A todas las personas de mi familia que me apoyaron y creyeron en mí día a día. A mis amigos de la Universidad y de Sogamoso por ser personas importantes y únicas. TABLA DE CONTENIDO 1. Introducción...................................................................................................... 1 2. Marco teórico.................................................................................................... 3 2.1. Importancia y problemática del nitrógeno en la agricultura............................. 3 2.2. La rizosfera...................................................................................................... 3 2.3. Microorganismos fijadores de nitrógeno atmosférico (diazótrofos)................. 5 2.3.1. Fijación biológica de nitrógeno (FBN).......................................................... 5 2.3.2. Microorganismos diazotróficos simbióticos.................................................. 7 2.3.3. Microorganismos diazotróficos asimbióticos................................................ 9 2.5. Familia Azotobacteraceae............................................................................... 11 2.6. Generalidades del género Azotobacter spp.................................................... 12 2.6.1. Azotobacter chroococcum............................................................................ 16 2.6.2. Azotobacter vinelandii................................................................................. 17 2.7. Técnicas moleculares para la caracterización e identificación de bacterias diazotrófas.............................................................................................................. 18 2.7.1. Aplicaciones del DNA ribosomal 16S y su utilización en filogenia y taxonomía............................................................................................................... 19 2.7.2. Análisis de restricción del DNA ribosomal 16S amplificado (ARDRA)................................................................................................................. 21 2.7.3. Caracterización, identificación y filogenia del género Azotobacter y otras bacterias diazótrofas mediante el análisis de restricción del RNA ribosomal 16S (ARDRA)................................................................................................................ 21 3. Formulación del problema y justificación...................................................... 25 4. Objetivos........................................................................................................... 27 4.1. Objetivo general.............................................................................................. 27 4.2. Objetivos específicos....................................................................................... 27 5. Metodología...................................................................................................... 28 5.1. Lugar de muestreo.......................................................................................... 28 5.2. Muestreo del suelo.......................................................................................... 28 5.3. Procesamiento de muestras............................................................................ 29 5.3.1. Medición de pH de las muestras de suelo.................................................... 29 5.3.2. Aislamiento primario..................................................................................... 29 5.4. Aislamiento secundario.................................................................................. 30 5.4.1. Conservación de cepas mediante la técnica de criopreservación en 30 glicerol al 50% (v/v)................................................................................................ 5.4.2. Pigmentación de los aislamientos................................................................ 30 5.4.3. Identificación Bioquímica….......................................................................... 31 5.4.4. Cepa control................................................................................................. 31 5.5. Extracción de DNA.......................................................................................... 32 5.6. Amplificación de DNA ribosomal 16S con iniciadores Y1 y Y3....................... 33 5.7. Análisis de restricción del DNA ribosomal 16S amplificado (ARDRA)............ 34 5.7.1. Perfil electroforético para el ARDRA............................................................ 34 5.7.2. Análisis filogenético...................................................................................... 35 5.8. Restricción virtual............................................................................................ 35 6. Resultados........................................................................................................ 36 6.1. Medición de pH de los cultivos muestreados.................................................. 36 6.2. Porcentaje de recuperación de bacterias diazótrofas aerobias asimbióticas.. 36 6.3. Identificación morfológica de bacterias diazótrofas aerobias asimbióticas............................................................................................................ 37 6.3.1. Morfología de las colonias............................................................................ 37 6.3.2. Morfología de las células.............................................................................. 37 6.3.3. Pigmentación................................................................................................ 38 6.4. Identificación bioquímica................................................................................. 39 6.5. Extracción de DNA.......................................................................................... 41 6.6. Amplificación del DNA ribosomal 16S con los primers Y1 y Y3...................... 42 6.7. Análisis de restricción del DNA ribosomal 16S (ARDRA)............................... 43 6.7.1. Análisis con la enzima AluI........................................................................... 43 6.7.2. Análisis con la enzima HpaII........................................................................ 44 6.7.3. Análisis con la enzima RsaI.......................................................................... 46 6.8. Cladograma generado del análisis con las tres enzimas................................ 48 6.9. Restricción virtual............................................................................................ 48 6.9.1. Análisis virtual con la enzima AluI................................................................ 50 6.9.2. Análisis virtual con la enzima HpaII.............................................................
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