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Maestra En Ciencias Biológicas UNIVERSIDAD MICHOACANA DE SAN NICOLÁS DE HIDALGO FACULTAD DE BIOLOGÍA Programa institucional de Maestría en Ciencias Biológicas “Identificación de genes asociados a la síntesis de magnetita en la tortuga negra Chelonia agassizii.” TESIS QUE PARA OBTENER EL GRADO DE: Maestra en Ciencias Biológicas PRESENTA: Biol. María Guadalupe Rodríguez Jiménez Directora de Tesis: Dra. Alma Lilia Fuentes Farías Co-director: Dr. Jesús Campos García Morelia, Michoacán. Marzo del 2014 1 AGRADECIMIENTOS A Dios por darme la dicha de tener una hermosa familia, a mis padres y hermanos por todo el apoyo incondicional durante todos estos años y por haber compartido conmigo momentos inolvidables. A la Dra. Alma Lilia Fuentes Farías y al Dr. Gabriel Gutiérrez Ospina por darme el espacio y la oportunidad de integrarme a su equipo de trabajo, por todo el apoyo brindado y el esfuerzo dedicado al proyecto durante todo este tiempo. Al Dr. Jesús Campos García por recibirme en su Laboratorio de Biotecnología Microbiana en el IIQB, por las horas incansables de trabajo y esfuerzo dedicado al proyecto. Al Dr. Alejandro Bravo Patiño y a la Dra. Esperanza Meléndez Herrera, por sus valiosos comentarios y sugerencias que sin duda enriquecieron de manera sustancial al trabajo. A la M.C. Alma Laura Díaz Pérez, por el apoyo brindado, sus valiosas aportaciones y sugerencias durante la fase experimental. Al Dr. Jesús Ramírez Santos, por las sugerencias y el apoyo técnico brindado durante el proyecto. Al Dr. Irvin Jácome y al Dr. Víctor Meza por el apoyo brindado en la fase de expresión de genes en el trabajo. A todos mis amigos, compañeros de laboratorio por su apoyo y compañía. ¡A TODOS MIL GRACIAS! 2 CONTENIDO LISTA DE FIGURAS .......................................................................................... 7 LISTA DE TABLAS ......................................................................................... 10 CAPITULO I. AMPLIFICACIÓN Y EXPRESIÓN DE GENES HOMOLOGOS DE LA BACTERIA, Magnetospirullum magneticum AMB-1 EN LA TORTUGA NEGRA Chelonia agassizii ............................................................................ 12 I. RESUMEN GENERAL ................................................................................. 12 II. SUMMARY ................................................................................................... 13 III. INTRODUCCIÓN GENERAL ...................................................................... 14 3.1. Transducción de la información magnética ambiental ............................... 14 3.2. Sintesis de magnetita ................................................................................ 20 IV. HIPÓTESIS DE INVESTIGACIÓN .............................................................. 25 V. OBJETIVOS ............................................................................................... 25 VI. MATERIALES Y METODOS ..................................................................... 26 6.1. Colecta de muestras ................................................................................. 26 6.2. Selección in silico de los genes candidatos a amplificar por PCR ............. 26 6.3. Diseño de oligonucleótidos degenerados .................................................. 28 6.4. Amplificación de los fragmentos de ADN por PCR .................................... 29 6.5. Electroforesis en gel de agarosa ............................................................... 29 3 6.6. Aislamiento y purificación de los productos de PCR ................................. 29 6.7. Ligación de los fragmentos de PCR y transformación de células de E. coli (JM101) ............................................................................................................ 29 6.8. Aislamiento de DNA plasmidico de bacterias ............................................ 30 6.9. Secuenciación de los fragmentos de PCR ................................................ 30 6.10. Análisis de las secuencias parciales obtenidas ....................................... 30 6.11. Análisis de similitud de las diferentes secuencias proteicas homólogas a los genes mam de bacteria, en la tortuga negra C. agassizii ................................. 31 6.12. Colecta de muestras de diferentes tejidos tortuga negra ........................ 32 6.13. Extracción y cuantificación de RNA mensajero ....................................... 32 6.14. Tratamiento de las muestras de RNAm con DNAsa I ............................. 32 6.15. Electroforesis en gel de agarosa ............................................................. 33 6.16. Ensayos de PCR en tiempo real cuantitativa (RT-qPCR)........................ 33 VII. RESULTADOS Y DISCUSIÓN .................................................................. 36 7.1. Amplificación de secuencias parciales en C. agassizii .............................. 36 A. Gen mamB ........................................................................................ 36 B. Gen mamK ........................................................................................ 40 C. Gen mamN ........................................................................................ 43 D. Gen mamO ........................................................................................ 47 E. Gen mamA ........................................................................................ 51 7.2. Análisis de similitud de genes mam de M. magneticum AMB-1, con el genoma de C. agassizii .................................................................................................. 55 4 A. Gen mamB ........................................................................................ 56 B. Gen mamK ........................................................................................ 57 C. Gen mamN ........................................................................................ 59 D. Gen mamO ........................................................................................ 60 E. Gen mamH ........................................................................................ 62 F. Gen mamM ........................................................................................ 63 G. Gen mamA ........................................................................................ 64 7.3. Expresión de genes por RT-qPCR ............................................................ 65 VIII. CONCLUSIONES ..................................................................................... 67 IX. PERSPECTIVAS ........................................................................................ 67 X. LITERATURA CITADA ................................................................................ 68 5 IX. ANEXOS ..................................................................................................... 72 ANEXO 1. mamB. Blastp de la secuencia obtenida para C. agassizii con la bacteria M. magneticum AMB-1 ............................................................. 72 ANEXO 2. mamB. Blastp de la secuencia obtenida para C. agassizii con el genoma de la tortuga C. mydas ............................................................. 74 ANEXO 3. mamK. Blastp de la secuencia obtenida para C. agassizii con la bacteria M. magneticum AMB-1 ............................................................. 80 ANEXO 4. mamK. Blastp de la secuencia obtenida para C. agassizii con el genoma de la tortuga C. mydas ............................................................. 86 ANEXO 5. mamN. Blastp de la secuencia obtenida para C. agassizii con la bacteria M. magneticum AMB-1 ............................................................. 87 ANEXO 6. mamN. Blastp de la secuencia obtenida para C. agassizii con el genoma de la tortuga C. mydas ............................................................. 89 ANEXO 7. MamO. Blastp de la secuencia obtenida para C. agassizii con la bacteria M. magneticum AMB-1 ............................................................. 95 ANEXO 8. MamO. Blastp de la secuencia obtenida para C. agassizii con el genoma de la tortuga C. mydas ............................................................. 96 ANEXO 9. MamA. Blastp de la secuencia obtenida para C. agassizii con el genoma de la tortuga C. mydas ........................................................... 102 ANEXO 10. Secuencias empleadas para el análisis de similitud de cada uno de los 7 genes mam de c. agassizii .............................................. 107 6 LISTA DE FIGURAS Figura 1. Esquemas de las rutas recorridas por las crías de tortuga Chelonia agassizii de cada uno de los grupos experimentales ....................................... 18 Figura 2. Diferentes áreas del cerebro responsivas a estímulos magnéticos mediante el uso de c-fos .................................................................................. 19 Figura 3. Respuestas electrofisiológicas evocadas ......................................... 19 Figura 4. Respuestas electromiográficas evocadas ........................................ 20 Figura 5. Identificación de cristales de magnetita biogénica en C. agassizii .. 20 Figura 6. Amplificación de los fragmentos obtenidos por PCR para el gen mamB de C. agassizii .................................................................................................. 37 Figura 7. Representación gráfica de la secuencia nucleotídica para el homólogo a mamB en C. agassizii ....................................................................................... 38 Figura 8. Representación gráfica de la secuencia en aminoácidos
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