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Variabilidad Genética De Los Genes P26 Y P10 En Aislamientos

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Variabilidad Genética De Los Genes P26 Y P10 En Aislamientos Variabilidad genética de los genes P26 y P10 en aislamientos colombianos de Potato yellow vein virus y análisis estructural de las proteínas en ortólogos de Crinivirus John Alexander Calderón Romero Universidad Nacional de Colombia Facultad de Ciencias Agrarias, Departamento Agronomía Bogotá, D.C. Colombia 2018 Variabilidad genética de los genes P26 y P10 en aislamientos colombianos de Potato yellow vein virus y análisis estructural de las proteínas en ortólogos de Crinivirus Jhon Alexander Calderón Romero Tesis para optar al título de: Magister en Ciencias Agrarias - con énfasis en Fitopatología Directora: MSc. Ángela María Vargas Berdugo Codirectora: Prof. Dr. Teresa de Mosquera Vásquez Línea de Investigación: Fitopatología Grupo de Investigación: Genética de Rasgos de Interés Agronómico Universidad Nacional de Colombia Facultad de Ciencias Agrarias, Departamento de Agronomía Bogotá, D.C., Colombia 2018 Dedicatoria Dedicado a toda mi familia y amig@s “Lo que sabemos es una gota de agua y lo que ignoramos es el océano” Isaac Newton Soy el médico de los cultivos y mis pacientes… son plantas. Agradecimientos A Dios por permitirme tener vida y salud A toda mi familia y núcleo familiar por ser la base y la fuerza que me motivó a seguir adelante A la Profesora Teresa Mosquera por hacer posible este sueño, por recibirme en su laboratorio y por todo el apoyo brindado A la profesora Ángela Vargas, por hacer posible este sueño, por su gran apoyo, por sus conocimientos y su constante entrega Al grupo de investigación de genética y rasgos complejos de interés agronómico por abrirme las puertas a su familia A la Universidad Nacional de Colombia por abrirme las puertas al mundo académico Presentación La investigación titulada Variabilidad genética de los genes P26 y P10 en aislamientos colombianos de Potato yellow vein virus y análisis estructural de las proteínas en ortólogos de Crinivirus se presenta en dos capítulos. La primera parte corresponde al resumen, summary, una introducción general del patosistema papa (Solanum tuberosum) y virus (Potato yellow vein virus), donde se muestran las principales características pues en cada uno de los artículos principales se trabajará su introducción. El capítulo uno corresponde al artículo “Variabilidad y recombinación de los genes P26 y P10 de Potato yellow vein virus (PYVV)” y el segundo capítulo corresponde al artículo “Patrones conservados de las proteínas P26 y P10 de Potato yellow vein virus (PYVV) con otros Crinivirus”. Al final de la tesis se presentan las conclusiones principales y las perspectivas para futuras investigaciones. 10 Variabilidad y estructura de los genes P26 y P10 de PYVV Contenido Resumen .........................................................................................................................12 Summary ........................................................................................................................13 1. Marco teórico ..........................................................................................................14 1.1 El patosistema papa- PYVV .............................................................................. 14 1.2 La familia Closteroviridae y las proteínas ortólogas en Crinivirus ......................... 16 1.3 Variabilidad en la familia Closteroviridae ........................................................... 17 1.4 Referencias ....................................................................................................... 19 Capítulo 1. Variabilidad y recombinación de los genes P26 y P10 en aislamientos colombianos de Potato yellow vein virus ....................................................................25 Abstract ....................................................................................................................... 27 1. Introducción .......................................................................................................... 28 2. Materiales y Métodos ........................................................................................... 30 2.1 Muestreo y material vegetal............................................................................30 2.2 Diseño de cebadores .........................................................................................31 2.3 Extracción de ARN total ......................................................................................31 2.4 Amplificación del gen de la proteína de la cápside de PYVV .........................32 2.5 Verificación de cebadores, RT-PCR y secuenciación ..........................................32 2.6 Preparación de secuencias de los genes P26 y P10 del virus PYVV ...........33 2.7 Diversidad y variabilidad de los genes P26 y P10 de PYVV ................................33 2.8 Eventos de recombinación en los genes P26 y P10 del virus PYVV .............34 3. Resultados ........................................................................................................... 34 3.1 Muestreo y material vegetal ................................................................................34 3.2 Diseño de cebadores para los genes P10 y P26 del virus PYVV ....................36 3.3 Secuencias de los genes P10 y P26 ...................................................................37 3.4 Diversidad de haplotipos en secuencias de aislamientos de los genes P26 y P10 del virus PYVV ..........................................................................................................39 42 3.5 Eventos de recombinación en los genes P10 y P26 del virus PYVV ...................43 3.6 Relaciones filogenéticas .....................................................................................43 3.7 Distancias genéticas entre secuencias de aislamientos de los genes P10 y P26 del virus PYVV ..........................................................................................................44 Variabilidad y estructura de los genes P26 y P10 de PYVV 11 3.8 Variabilidad, diversidad y test de neutralidad de los genes P10 y P26 del virus PYVV 48 4. Discusión ............................................................................................................. 50 5. 54 Conclusiones ................................................................................................................. 54 6. ................................................................................................................................ 55 Referencias ................................................................................................................ 55 Capítulo 2. Patrones conservados de las proteínas P26 y P10 de PYVV con otros Crinivirus ....................................................................................................................... 61 Resumen .................................................................................................................... 62 Abstract ...................................................................................................................... 63 1. Introducción ......................................................................................................... 64 2. Materiales y métodos ........................................................................................... 66 2.1 Análisis de secuencias y modelamiento ............................................................. 66 2.4 Estructura secundaria de las proteínas del grupo P10 y P26 de Crinivirus . 68 2.5 Estructura terciaria y validación de las proteínas P10 y P26 ortólogas de Crinivirus ................................................................................................................ 68 3. Resultados ............................................................................................................ 69 3.1 Alineamiento múltiple ......................................................................................... 69 3.2 Estructura primaria y modificaciones postraduccionales ..................................... 72 3.2.1 Perfiles de hidrofobicidad ................................................................................ 72 3.2.2 Modificaciones postraduccionales ................................................................... 75 3.3 Estructura secundaria de las proteínas ortólogas P10 y P26 de Crinivirus ......... 76 3.4 Topología transmembranal de las proteínas ortólogas de P10 y P26 de Crinivirus79 3.5 Estructura terciaria de las proteínas ortólogas P10 y P26 de Crinivirus .............. 80 4. Discusión ............................................................................................................. 84 5. Conclusiones .......................................................................................................... 86 La función de la proteína P26 de PYVV puede ser la inducción de depósitos de plasmalema en las células del floema, pués conserva la misma estructura secundaria y terciaria que los ortólogos en otras especies que han sido demostrada experimentalmente, de igual forma la función de la proteína P10 debería ser similar a la de todos los crinivirus pero se debe entrar a determinar experimentalmente cual es esta. ............................................................................................................................ 86 6. Referencias ......................................................................................................... 87 12 Variabilidad y estructura de los genes P26 y P10 de PYVV Resumen Potato yellow vein virus es un virus reemergente, cuarentenario,
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