Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro

Colombia es el primer productor mundial de clavel y el patógeno, Fusarium oxysporum f.sp. dianthi (Fod) causa grandes pérdidas en el cultivo comercial de esta planta. La producción de variedades resistentes al hongo es una de las formas más efectivas para controlar los daños producidos por el parási...

Full description

Autores:
Gomez Corredor, William Andres
Tipo de recurso:
Trabajo de grado de pregrado
Fecha de publicación:
2021
Institución:
Universidad Militar Nueva Granada
Repositorio:
Repositorio UMNG
Idioma:
spa
OAI Identifier:
oai:repository.unimilitar.edu.co:10654/41286
Acceso en línea:
http://hdl.handle.net/10654/41286
Palabra clave:
Plant resistance
Dianthus caryophyllus
Defense response
RT-qPCR
Differential expression
Orthologous genes
DIANTHUS CARYOPHYLLUS
RESISTENCIA A LA ENFERMEDAD
FUSARIUM OXYSPORUM
IN VITRO
Resistencia vegetal
Dianthus caryophyllus
Respuesta de defensa
RT-qPCR
Expresión diferencial
Genes ortólogos
Rights
openAccess
License
http://creativecommons.org/licenses/by-nc-nd/4.0/
id UNIMILTAR2_a4ae69cd1459b5e7dfe499ae0f08377a
oai_identifier_str oai:repository.unimilitar.edu.co:10654/41286
network_acronym_str UNIMILTAR2
network_name_str Repositorio UMNG
repository_id_str
dc.title.spa.fl_str_mv Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
dc.title.translated.spa.fl_str_mv Study of the variation of orthologic gene expression in resistant and susceptible varieties of Dianthus caryophyllus elictified with Fusarium oxysporum f.sp. dianthi under in vitro conditions
title Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
spellingShingle Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
Plant resistance
Dianthus caryophyllus
Defense response
RT-qPCR
Differential expression
Orthologous genes
DIANTHUS CARYOPHYLLUS
RESISTENCIA A LA ENFERMEDAD
FUSARIUM OXYSPORUM
IN VITRO
Resistencia vegetal
Dianthus caryophyllus
Respuesta de defensa
RT-qPCR
Expresión diferencial
Genes ortólogos
title_short Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
title_full Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
title_fullStr Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
title_full_unstemmed Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
title_sort Estudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitro
dc.creator.fl_str_mv Gomez Corredor, William Andres
dc.contributor.advisor.none.fl_str_mv Filgueira Duarte, Juan Jose
dc.contributor.author.none.fl_str_mv Gomez Corredor, William Andres
dc.subject.keywords.spa.fl_str_mv Plant resistance
Dianthus caryophyllus
Defense response
RT-qPCR
Differential expression
Orthologous genes
topic Plant resistance
Dianthus caryophyllus
Defense response
RT-qPCR
Differential expression
Orthologous genes
DIANTHUS CARYOPHYLLUS
RESISTENCIA A LA ENFERMEDAD
FUSARIUM OXYSPORUM
IN VITRO
Resistencia vegetal
Dianthus caryophyllus
Respuesta de defensa
RT-qPCR
Expresión diferencial
Genes ortólogos
dc.subject.agrovoc.spa.fl_str_mv DIANTHUS CARYOPHYLLUS
RESISTENCIA A LA ENFERMEDAD
FUSARIUM OXYSPORUM
IN VITRO
dc.subject.proposal.spa.fl_str_mv Resistencia vegetal
Dianthus caryophyllus
Respuesta de defensa
RT-qPCR
Expresión diferencial
Genes ortólogos
description Colombia es el primer productor mundial de clavel y el patógeno, Fusarium oxysporum f.sp. dianthi (Fod) causa grandes pérdidas en el cultivo comercial de esta planta. La producción de variedades resistentes al hongo es una de las formas más efectivas para controlar los daños producidos por el parásito. A pesar de que se han realizado trabajos que han descrito el desarrollo de la enfermedad en clavel causadas por Fod, así como estudios sobre los cambios histológicos y bioquímicos en las variedades resistentes y susceptibles, los genes que gobiernan la respuesta de defensa en contra del ataque del patógeno no se han caracterizado aún. Sabemos por trabajos anteriores en nuestro grupo de investigación, que la resistencia del clavel a Fod esta mediada por tres pares de genes autosómicos con herencia simple y aditiva, pero es necesario reconocer estos genes de tal forma que se pueda utilizar este conocimiento para conducir apropiadamente los programas de mejoramiento del clavel comercial. En el presente trabajo se obtuvo el material biológico, usando células indiferenciadas de clavel y Fod cepa 2019, evaluado los oligos por amplificación por PCR en ADN genómico, posteriormente se evaluaron 22 parejas de oligos identificados en trabajos previos como housekeeping, genes relacionados con resistencia a parásitos y genes que codifican a proteínas relacionadas con patogenicidad mediante RT-qPCR, relacionados con la respuesta de defensa a Fod en las variedades resistentes UMNG395 y UMNGF1B2 y variedades susceptibles UMNGSH2 y UMNG6515 del programa de mejoramiento del clavel de la UMNG. Demostrando que la expresión unos genes en fenotipos resistentes no está asociado a la resistencia, sino que un conjunto de genes son parte de un metabolismo relacionado con la respuesta de defensa frente al patógeno, que abarca procesos de señalización, regulación, inducción, transcripción de genes.
publishDate 2021
dc.date.issued.none.fl_str_mv 2021-12-14
dc.date.accessioned.none.fl_str_mv 2022-09-02T14:25:30Z
dc.date.available.none.fl_str_mv 2022-09-02T14:25:30Z
dc.type.local.spa.fl_str_mv Tesis/Trabajo de grado - Monografía - Pregrado
dc.type.driver.spa.fl_str_mv info:eu-repo/semantics/bachelorThesis
dc.type.coar.*.fl_str_mv http://purl.org/coar/resource_type/c_7a1f
format http://purl.org/coar/resource_type/c_7a1f
dc.identifier.uri.none.fl_str_mv http://hdl.handle.net/10654/41286
dc.identifier.instname.spa.fl_str_mv instname:Universidad Militar Nueva Granada
dc.identifier.reponame.spa.fl_str_mv reponame:Repositorio Institucional Universidad Militar Nueva Granada
dc.identifier.repourl.spa.fl_str_mv repourl:https://repository.unimilitar.edu.co
url http://hdl.handle.net/10654/41286
identifier_str_mv instname:Universidad Militar Nueva Granada
reponame:Repositorio Institucional Universidad Militar Nueva Granada
repourl:https://repository.unimilitar.edu.co
dc.language.iso.spa.fl_str_mv spa
language spa
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spelling Filgueira Duarte, Juan JoseGomez Corredor, William AndresBiólogo2022-09-02T14:25:30Z2022-09-02T14:25:30Z2021-12-14http://hdl.handle.net/10654/41286instname:Universidad Militar Nueva Granadareponame:Repositorio Institucional Universidad Militar Nueva Granadarepourl:https://repository.unimilitar.edu.coColombia es el primer productor mundial de clavel y el patógeno, Fusarium oxysporum f.sp. dianthi (Fod) causa grandes pérdidas en el cultivo comercial de esta planta. La producción de variedades resistentes al hongo es una de las formas más efectivas para controlar los daños producidos por el parásito. A pesar de que se han realizado trabajos que han descrito el desarrollo de la enfermedad en clavel causadas por Fod, así como estudios sobre los cambios histológicos y bioquímicos en las variedades resistentes y susceptibles, los genes que gobiernan la respuesta de defensa en contra del ataque del patógeno no se han caracterizado aún. Sabemos por trabajos anteriores en nuestro grupo de investigación, que la resistencia del clavel a Fod esta mediada por tres pares de genes autosómicos con herencia simple y aditiva, pero es necesario reconocer estos genes de tal forma que se pueda utilizar este conocimiento para conducir apropiadamente los programas de mejoramiento del clavel comercial. En el presente trabajo se obtuvo el material biológico, usando células indiferenciadas de clavel y Fod cepa 2019, evaluado los oligos por amplificación por PCR en ADN genómico, posteriormente se evaluaron 22 parejas de oligos identificados en trabajos previos como housekeeping, genes relacionados con resistencia a parásitos y genes que codifican a proteínas relacionadas con patogenicidad mediante RT-qPCR, relacionados con la respuesta de defensa a Fod en las variedades resistentes UMNG395 y UMNGF1B2 y variedades susceptibles UMNGSH2 y UMNG6515 del programa de mejoramiento del clavel de la UMNG. Demostrando que la expresión unos genes en fenotipos resistentes no está asociado a la resistencia, sino que un conjunto de genes son parte de un metabolismo relacionado con la respuesta de defensa frente al patógeno, que abarca procesos de señalización, regulación, inducción, transcripción de genes.CAPÍTULO 1: REVISIÓN BIBLIOGRÁFICA 18 1. Clavel (Dianthus caryophyllus L.) 18 1.1. Importancia económica del clavel 19 1.2. Mejoramiento genético del clavel 20 2. Fusarium oxysporum 22 2.1. Fusarium oxysporum f.sp. dianthi (Fod) 23 2.2. Problemática causada por Fusarium oxysporum 23 2.3. Enfermedad causada por Fusarium oxysporum 24 2.4. Implicaciones en el cultivo de clavel 25 3. Manejo Enfermedad 27 3.1. Manejo tradicional de la enfermedad 27 3.2. Manejo biotecnológico de la enfermedad 28 4. Mecanismos de defensa Planta – Patógeno 29 4.1. Resistencia genética a Fusarium oxysporum 31 4.2. Mecanismos de regulación de la defensa vegetal contra factores de estrés 33 4.3. Algunas rutas de defensa en plantas, asociadas a metabolitos secundarios y dirigidas contra fitopatógenos 36 5. Uso de herramientas moleculares 37 CAPÍTULO II. ESCRITO TRABAJO DE GRADO 38 1. RESUMEN - ABSTRACT 39 2. INTRODUCCIÓN 39 3. OBJETIVOS 41 3.1. Objetivo general 42 3.2. Objetivos Específicos 42 4. MATERIALES Y MÉTODOS 42 4.1. Obtención del Material Biológico. 43 4.2. Propagación in vitro de células indiferenciadas (CIS) de variedades Resistentes y Susceptibles 43 4.3. Obtención de Fusarium oxysporum a partir de muestras de plantas infectadas. 44 4.4. Obtención de cultivos monospóricos de Fusarium oxysporum 44 4.5. Ensayo de reinfección y aislamiento de Fusarium oxysporum f.sp. dianthi (Fod) 45 4.6. Pruebas de Elicitación 45 4.7. Extracción y cuantificación de ADN de CIS 46 4.8. Extracción y cuantificación de ARN total de CIS. 46 4.9. Amplificación por PCR 47 4.10. RT-qPCR 48 4.10.1. Genes evaluados 49 4.11. Análisis de resultados de expresión 50 CAPÍTULO 3: RESULTADOS Y DISCUSIÓN 52 1. Obtención material biológico 52 1.1. Propagación in vitro de células indiferenciadas (CIS) de variedades resistentes y susceptibles 52 1.2. Obtención de Fusarium oxysporum a partir de muestras de plantas infectadas. 53 1.3. Caracterización morfológica 53 1.4. Obtención de cultivos monospóricos de Fusarium oxysporum 54 1.5. Ensayo de reinfección y aislamiento de Fusarium oxysporum f.sp. dianthi (Fod) 55 1.6. Ensayo de Elicitación 56 2. Evaluación material genético 58 2.1. Extracción y Cuantificación de ADN de CIS. 58 2.2. Amplificación por PCR de secuencias génicas 59 2.2.1. PCR de genes “Housekeeping” 59 2.2.2. PCR de genes asociados a la resistencia a parásitos 61 2.2.3. PCR de genes asociados con proteínas relacionadas con patogenicidad 63 2.3. Extracción y cuantificación de ARN total de CIS 67 3. Análisis de expresión 68 3.1. Genes “Housekeeping” 68 3.1.1. Alcohol deshidrogenasa 69 3.1.2. Arginina descarboxilasa 71 3.1.3. Tetrahidroxichalcona 2-glucosiltransferasa 72 3.1.4. “Ethylene Insensitive 3-like 2” 74 3.2. Genes relacionados con la resistencia a parásitos 76 3.2.1. Antranilato N-hidroxicinamoil / Benzoiltransferasa 77 3.2.2. Gen receptor de etileno 78 3.2.3. Flavonol 3-Glucosiltransferasa 79 3.2.4. Fenilalanina amonio liasa 81 3.2.5. Antocianidin sintasa 83 3.3. Genes relacionados con proteínas de patogenicidad 85 3.3.1. β-(1 3)-D-Glucanasa 86 3.3.2. Quitinasa 88 3.3.3. Hidrolasas de glucósido de β-glicosidasa 90 3.3.4. Aldosa 1-epimerasa 91 3.3.5. “Thaumatin Like Protein” 93 3.3.6. β- amilasa 95 3.3.7. Homólogo a peroxidasas 97 3.3.8. Catalasa 100 3.3.9. Malato deshidrogenasa 102 3.3.10. Vía de señalización mediada por ácido jasmonico 104 3.3.11. Proteína inhibidora de poligalacturonasas 106 3.3.12. Vía de señalización mediada por ácido salicílico 108 3.3.13. “Receptor- like Kinase” 111 4. CONCLUSIONES 113 5. BIBLIOGRAFÍA 114 6. ANEXOS 141 6.1. Gráficas análisis de expresión RT-qPCR 141 6.1.1. Gráficas RT-qPCR genes “Housekeeping” 141 6.1.2. Gráficas de RT-qPCR de Genes relacionados con resistencia a parásitos 146 6.1.3. Genes relacionados con proteínas de patogenicidad 151 6.1.4. Gráficas ecuación lineal genes 162Colombia is the world's leading producer of carnation and the pathogen, Fusarium oxysporum f.sp. dianthi (Fod) causes great losses in the commercial cultivation of this plant. The production of varieties resistant to the fungus is one of the most effective ways to control the damage caused by the parasite. Although work has been done describing the development of the disease in carnation caused by Fod, as well as studies on the histological and biochemical changes in resistant and susceptible varieties, the genes that govern the defense response against the pathogen attack have not yet been characterized. We know from previous work in our research group that carnation resistance to Fod is mediated by three pairs of autosomal genes with simple and additive inheritance, but it is necessary to recognize these genes so that this knowledge can be used to properly conduct commercial carnation breeding programs. In the present work, biological material was obtained, using undifferentiated cells of carnation and Fod strain 2019, evaluated the oligos by PCR amplification in genomic DNA, then 22 pairs of oligos identified in previous works as housekeeping were evaluated, genes related to parasite resistance and genes encoding proteins related to pathogenicity were evaluated by RT-qPCR, related to the defense response to Fod in resistant varieties UMNG395 and UMNGF1B2 and susceptible varieties UMNGSH2 and UMNG6515 of the UMNG carnation breeding program. It shows that the expression of some genes in resistant phenotypes is not associated with resistance, but that a set of genes are part of metabolism related to the defense response against the pathogen, which includes processes of signaling, regulation, induction, and transcription of genes.Pregradoapplicaction/pdfspahttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Attribution-NonCommercial-NoDerivatives 4.0 InternationalAcceso abiertoEstudio de la variación de la expresión de genes ortólogos en variedades resistentes y susceptibles de Dianthus caryophyllus elicitadas con Fusarium oxysporum f.sp. dianthi en condiciones in vitroStudy of the variation of orthologic gene expression in resistant and susceptible varieties of Dianthus caryophyllus elictified with Fusarium oxysporum f.sp. dianthi under in vitro conditionsTesis/Trabajo de grado - Monografía - Pregradoinfo:eu-repo/semantics/bachelorThesishttp://purl.org/coar/resource_type/c_7a1fPlant resistanceDianthus caryophyllusDefense responseRT-qPCRDifferential expressionOrthologous genesDIANTHUS CARYOPHYLLUSRESISTENCIA A LA ENFERMEDADFUSARIUM OXYSPORUMIN VITROResistencia vegetalDianthus caryophyllusRespuesta de defensaRT-qPCRExpresión diferencialGenes ortólogosBiología AplicadaFacultad de Ciencias BásicasUniversidad Militar Nueva Granada1. 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Disponible en: https://doi.org/10.21273/JASHS.126.1.13Campus UMNGLICENSElicense.txtlicense.txttext/plain; charset=utf-83420http://repository.unimilitar.edu.co/bitstream/10654/41286/2/license.txta609d7e369577f685ce98c66b903b91bMD52ORIGINALGomezCorredorWilliamAndres2021.pdfGomezCorredorWilliamAndres2021.pdfTrabajo de gradoapplication/pdf4313248http://repository.unimilitar.edu.co/bitstream/10654/41286/1/GomezCorredorWilliamAndres2021.pdf1307ec3fb4c1ff2087743422f2c36beaMD5110654/41286oai:repository.unimilitar.edu.co:10654/412862022-09-02 09:25:31.561Repositorio Institucional 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