Obtención de una mutante insercional para el gen Cas9 en el hongo fitopatógeno Botrytis cinerea para su utilización como herramienta de edición génica implementando el sistema CRISPR/Cas9
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Fecha
2020
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
Botrytis cinerea es un hongo patógeno y necrotrófico de plantas de alto
interés comercial. Denominado comúnmente como “moho gris”, este produce la
pudrición de la parte aérea de las plantas y la podredumbre de vegetales, frutas
y flores después de ser cosechados, generando grandes pérdidas económicas
en Chile y el mundo. B. cinerea presenta una extraordinaria variabilidad y
plasticidad genética, convirtiéndolo en un modelo de estudio clave en la relación
planta-patógeno. Actualmente, existe una alta demanda por el desarrollo de
técnicas más simples y versátiles en comparación a las disponibles para la
manipulación genética de este y otros hongos filamentosos. Por esta razón, el
sistema CRISPR/Cas9, Claustered Regularly Interspaced Short Palindromic
Reapeats (repeticiones palindrómicas cortas, agrupadas y regularmente
interespaciadas) es un foco de interés para este hongo y otros organismos
patógenos. Este mecanismo inmune de bacterias se ha convertido, en los últimos
años, en una potente herramienta de edición de genes, formado por dos
componentes, la nucleasa Cas9 y un guía de RNA. Esta ribonucleoproteína actúa
como una tijera molecular que permite la introducción de roturas bicatenarias
específicas de DNA, facilitando una eficaz selección/edición de genes. En esta
tesis, se generó una mutante insercional para el gen Cas9 en el locus del gen
Ku70 en B. cinerea. Las mutantes obtenidas fueron genotipificadas mediante
PCR. Resultados preliminares muestran un fenotipo de crecimiento similar a la
cepa B05.10.
Botrytis cinerea is a pathogenic and necrotrophic fungus of plants of high commercial interest. Commonly known as "gray mold," it causes the rotting of the aerial part of plants and vegetables, as well as fruits and flowers in postharvest conditions, generating large economic losses in Chile and worldwide. B. cinerea presents an extraordinary variability and genetic plasticity, making it a great biological model to study the plant-pathogen interaction. Currently, there is a need for the development of versatile and simpler techniques for its genetic manipulation, as well as in other filamentous fungi. For these reasons, the CRISPR / Cas9 system (short palindromic repeats, grouped and regularly interspaced) represents a great alternative. Therefore, it is a matter of interest for this fungus and other pathogenic organisms. CRISPR was described as a bacterial immune mechanism and has become a powerful gene-editing tool. It consists of two components, the Cas9 nuclease, and an RNA guide. This system acts as a molecular scissor that allows the introduction of specific double-stranded DNA breaks, facilitating an efficient gene selection/editing. In this thesis work, a knocking mutant for the Cas9 gene was generated in the locus of the ku70 gene in B. cinerea. The obtained mutants were genotyped by PCR. Preliminary results showed a growth phenotype similar to the B05.10 wildtype strain
Botrytis cinerea is a pathogenic and necrotrophic fungus of plants of high commercial interest. Commonly known as "gray mold," it causes the rotting of the aerial part of plants and vegetables, as well as fruits and flowers in postharvest conditions, generating large economic losses in Chile and worldwide. B. cinerea presents an extraordinary variability and genetic plasticity, making it a great biological model to study the plant-pathogen interaction. Currently, there is a need for the development of versatile and simpler techniques for its genetic manipulation, as well as in other filamentous fungi. For these reasons, the CRISPR / Cas9 system (short palindromic repeats, grouped and regularly interspaced) represents a great alternative. Therefore, it is a matter of interest for this fungus and other pathogenic organisms. CRISPR was described as a bacterial immune mechanism and has become a powerful gene-editing tool. It consists of two components, the Cas9 nuclease, and an RNA guide. This system acts as a molecular scissor that allows the introduction of specific double-stranded DNA breaks, facilitating an efficient gene selection/editing. In this thesis work, a knocking mutant for the Cas9 gene was generated in the locus of the ku70 gene in B. cinerea. The obtained mutants were genotyped by PCR. Preliminary results showed a growth phenotype similar to the B05.10 wildtype strain
Notas
Tesis (Licenciado en Biología)
Palabras clave
Botrytis Cinerea, Genética, Hongos Fitopatógenos