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Facultad De Ciencias Forestales Y Del Ambiente UNIVERSIDAD NACIONAL DEL CENTRO DEL PERÚ Facultad de Ciencias Forestales y del Ambiente TESIS “CARACTERIZACION MORFOLOGICA Y MOLECULAR DE SEIS ESPECIES DEL GENERO Eucalyptus” Presentada por: Bach. OCHOA MONTANO, María Estela Bach. SALINAS ANAYA, Dayana Carmen PARA OPTAR EL TÍTULO PROFESIONAL DE INGENIERA FORESTAL Y AMBIENTAL HUANCAYO – PERÚ 2009 1 ASESORA Ing. M. Sc. Edith, Orellana Mendoza 2 DEDICATORIA Con todo mi amor y cariño. A ti Dios me diste la oportunidad de vivir y de regalarme a mi padre Saturnino y a mis hermanos. Quiero dedicar este trabajo a mi Madre adorada, Lupe por hacerme surgir y salir adelante demostrándome siempre valentía y entereza, a mi Mamita Carmen y a mi Papito Juan por darme todo el amor del mundo y a las personas que siempre estuvieron conmigo en las buenas y en las malas, Juan Carlos por su amor incondicional, Alfonso por su amistad sin fronteras, Nicolás por sus sabios consejos. Dana 3 AGRADECIMIENTOS A la Ing. M.Sc Edith Orellana Mendoza, por su asesoramiento y apoyo brindado en la ejecución y culminación de la presente tesis. Al Ing. Nicolás Román Cabello, investigador en Biotecnología e Ingeniería Genética, miembro del directorio del Instituto de Biotecnología de la Universidad Nacional del Centro del Perú, y Coordinador del Proyecto “Estudio de la Biomasa para Mejorar la Calidad del Suelo” del Fondo Internacional de Ciencia y Tecnología (FINCYT). Al M.Sc Gilberto Gamarra Sánchez, especialista en Fitomejoramiento y Docente de la Facultad de Agronomía – UNCP, por el apoyo en el análisis de los resultados del proyecto. Al Lic. Fisher Huaraca Meza, especialista en Fitomejoramiento de la Universidad Nacional Agraria La Molina de la Unidad del Centro de Investigación Biotecnológica – Lima, por el apoyo en la caracterización bioquímica. Al Biólogo Luis Gutiérrez Walhoff, del Centro Internacional de la Papa – Lima, especialista en análisis electroforéticos con DNA y PROTEINAS, por el apoyo en el uso de reactivos electroforéticos. Al Ing. Nicolás Huaringa Miranda, encargado del Vivero Forestal del Mantaro de la UNCP, por todas las facilidades brindadas durante la permanencia en la estación. Al laboratorio del Instituto de Biotecnología de la Universidad Nacional del Centro del Perú por brindarnos sus instalaciones y facilitarnos el Programa NTSYS pc Versión 2.10 y a sus asistentes el Ing. David García Ventocilla, Ing. Luis Ángel, Iparraguirre de la Cruz y a la Ing. Gloria Mamani Gamarra por su apoyo incondicional. 4 ÍNDICE Contenido Pág. RESUMEN I. INTRODUCCIÓN ...................................................................................... 1 II. REVISIÓN DE LITERATURA ................................................................... 3 2.1. ANTECEDENTES……………………………………………………….… ........ 3 2.2. BASES TEÓRICAS Y CIENTÍFICAS .................................................... 12 2.2.1. Electroforesis………………………………….…………...……… .... 12 2.2.2. Características de la electroforesis en geles de poliacrilamida ... 16 2.2.3. La porosidad del gel…………………………………………………. ..................................... 18 2.2.4. Tinción rápida……………………………………..……………….. ......................... 23 2.2.5. Tiempo para el teñido……………………………………………….. .................................. 23 2.2.6. Peso molecular (PM) de las proteínas…………...…….………… ........................................................ 26 2.3. DESCRIPCIÓN BOTÁNICA, DISTRIBUCIÓN Y TAXONOMÍA DE LAS ESPECIES ESTUDIADAS DEL EUCALIPTO .............................. 27 2.3.1. Eucalyptus ................................................................................... 27 2.3.2. Eucalyptus nitens ........................................................................ 27 2.3.3. Eucalyptus glóbulus Labill ........................................................... 28 2.3.4. Eucalyptus viminalis Labill ........................................................... 29 2.3.5. Eucalyptus maidenii F.Muell ....................................................... 30 2.3.6. Eucalyptus cinerea F.Muell. ex ................................................... 31 2.3.7 Eucalyptus gunnii Hook.f .............................................................. 31 2.3.8. Clasificación taxonómica del Eucalyptus .................................... 32 2.3.9. Información Química .................................................................... 32 5 2.4. RECURSOS GENETICOS .................................................................... 33 2.4.1. Estados del descriptor y tipos de datos ............................................. 36 2.5. PROTEÍNAS ......................................................................................... 37 2.5.1. Estructura de las proteínas ................................................................ 38 a) Estructura primaria .............................................................................. 38 b) Estructura secundaria .......................................................................... 39 c) Estructura terciaria ............................................................................... 40 d) Estructura cuaternaria ......................................................................... 41 e) Desnaturalización de proteínas ........................................................... 41 2.6. MARCADORES MOLECULARES ........................................................ 45 III. MATERIAL Y MÉTODO .......................................................................... 46 3.1. DESCRIPCIÓN DEL ÁREA DE ESTUDIO............................................ 46 3.1.1. Ubicación ........................................................................................... 46 3.1.2. Clima y Ecología ................................................................................ 47 3.2. MATERIALES Y EQUIPOS ................................................................... 47 3.2.1. Materiales de Campo ......................................................................... 47 3.2.2. Materiales de Laboratorio .................................................................. 48 3.2.3. Equipos de laboratorio ....................................................................... 49 3.2.4. Reactivos e Insumos .......................................................................... 50 3.3.4.1.Estandares de Masa molecular de Proteínas ........................ 51 3.2.5. Equipos de Campo ............................................................................ 51 3.2.6. Materiales y Equipos de Gabinete ..................................................... 51 3.3. METODOLOGIA ................................................................................... 51 3.3.1. Diseño Metodológico ......................................................................... 51 3.3.2. Población y Muestra .......................................................................... 53 3.3.3. Procedimiento Metodológico .............................................................. 53 3.3.3.1 Fase pre campo.................................................................... 53 a) Recopilación de información básica ................................. 53 3.3.3.2 Fase de campo ..................................................................... 53 a) Identificación y selección de las especies de Eucalyptus .............. 53 b) Colecta de semillas ....................................................................... 54 6 c) Descripción morfológica ................................................................ 54 3.3.3.3 Fase laboratorio.................................................................... 58 a) Preparación de la muestra ............................................................ 58 b) Preparación de reactivos ............................................................... 59 c) Desnaturalización de muestra ....................................................... 62 d) Preparación de los geles de poliacrilamida (PAGE)…………… .... 62 e) Ensamblaje de la cámara de electroforesis……………………… ..................................................... 65 f) Polimerización del gel………………………………..……………… ................................... 65 g)Tiempo de corrida…………………………………………………… ........................ 70 h)Tinción de los geles de poliacrilamida con azul de comassie ........ 71 i) Coloración y decoloración del gel………………………………… .... 71 j) Almacenamiento de los geles de poliacrilamida………………… .... 72 k) Preparación del marcador de pesos moleculares……………… .... 72 l) Determinación de los pesos moleculares de las proteínas en estudio ......................................................................................... 73 3.4. ANÁLISIS DE DATOS .......................................................................... 75 3.4.1. Datos de la caracterización morfológica ............................................ 75 3.4.2. Análisis de datos moleculares ............................................................ 75 IV. RESULTADOS.. ..................................................................................... 77 V. DISCUSIONES ........................................................................................ 93 VI. CONCLUSIONES ................................................................................... 101 VII. RECOMENDACIONES ......................................................................... 103 VII. BIBLIOGRAFÍA ...................................................................................... 104 ANEXOS 7 Lista de Tablas Tabla 1: Tamaño aproximado de algunas proteínas ................................... 19 Tabla 2: Descriptor
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