dc.contributor.advisor | Valencia-Jimenez, Arnubio | |
dc.contributor.author | Villegas-Estrada, Bernardo | |
dc.date.accessioned | 2022-07-15T16:33:17Z | |
dc.date.available | 2036-07-01 | |
dc.date.available | 2022-07-15T16:33:17Z | |
dc.date.issued | 2022-07-15 | |
dc.identifier.uri | https://repositorio.ucaldas.edu.co/handle/ucaldas/17836 | |
dc.description | Ilustraciones, gráficas | spa |
dc.description.abstract | spa:El silenciamiento génico postranscripcional (PTGS) es un mecanismo de defensa vegetal conservado evolutivamente contra los virus. Este trabajo tuvo como objetivos probar una construcción de dsDNA (77 pb) como plantilla para la producción in vitro de ARN de horquilla pequeña artificial (shRNA) derivado de virus, además de evaluar tanto estos como RNAs de doble cadena derivados de virus (dsRNA), también producidos in vitro, su potencial para desencadenar el mecanismo de ARNi en plantas de Nicotiana benthamiana contra CMV después de su infiltración foliar. Este enfoque permitió la producción de cantidades significativas de shRNA (60-mers) y de dsRNA (492-508-mers) de forma rápida y sencilla. El silenciamiento de los genes de CMV provocado por shRNA se confirmó mediante la reacción en cadena de la polimerasa (PCR), ensayos basados en inmunología y PCR en tiempo real (qPCR), mientras que el silenciamiento de los genes provocado por los dsRNA fue confirmado preliminarmente por PCR, Dot-blot y RT-qPCR. Los niveles más altos de silenciamiento génico por la mezcla de shRNAs se registraron para los ARNm que codifican la proteína de replicación (ORF1a), el supresor viral del silenciamiento de ARN (ORF2b) y la proteína de la cápside (ORF3b) con 98, 94 y 70 % del silenciamiento total de la transcripción, respectivamente, al igual que para la mezcla de dsRNAs cuyos niveles de silenciamiento fueron de 78 %, 65 % y 72 %, respectivamente, para los genes ORF1, ORF2a y ORF3b. El protocolo proporciona una alternativa a la producción de shRNA significativos y la aplicación tanto de estos como de dsRNAs que pueden desencadenar eficazmente el mecanismo de RNAi contra CMV. | spa |
dc.description.abstract | eng:Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs), to evaluating these and virus-derived double-stranded RNAs (dsRNA), also produced in vitro, their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed the production of significant amounts of shRNAs (60-mers) and dsRNA (492-508-mers) quickly and easily. ShRNA-induced silencing of CMV genes was confirmed by polymerase chain reaction (PCR), Immunological-based assays, and Real-Time PCR (qPCR) while dsRNA-induced gene silencing was confirmed preliminarily by PCR, Dot-blot and qPCR. The highest levels of gene silencing by the mixture of shRNAs were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b) with 98, 94, and 70 % of total transcript silencing, respectively. Similar results were obtained for the mixture of dsRNAs whose silencing levels were 78%, 65% and 72%, respectively for the genes ORF1, ORF2a and ORF3b. The protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV as well as dsRNAs. | eng |
dc.description.tableofcontents | AGRADECIMIENTOS/vi DEDICATORIA /vii LISTA DE FIGURAS/..viii LISTA DE TABLAS /.ix LISTA DE ANEXOS/.x Nota al lector./.xi Pregunta de investigación/ Objetivos/ Objetivo general/ Objetivos específicos:/ Descripción del trabajo: / CAPÍTULO I: INTRODUCCIÓN/Bibliografía / CAPÍTULO II: PRIMER ARTÍCULO / Evaluación de métodos para la inoculación y diagnóstico del virus del mosaico del pepino (CMV)*/ MÉTODO/ Inoculación del CMV / Detección del CMV en plantas inoculadas/ Sintomatología/ Serología / Moleculares/RT-PCR / RESULTADOS/ Métodos de detección del CMV/ Detección viral por sintomatología/ Detección viral mediante ImmunoStrip®/ Detección por RT-PCR/ CONCLUSIONES/ REFERENCIAS/ CAPÍTULO III: SEGUNDO ARTÍCULO / Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus / 1. Introduction / 2. Results / 2.1. Synthesis, quality, and yield of shRNAs/ v 2.2. Primer efficiency and specificity/ 2.3. Effect of virus-derived artificial shRNAs on CMV infection/ 2.4. Immunological-based assays for specific detection of CMV/ 2.5. PCR and qPCR / 3. Discussion / 4. Materials and Methods/ 4.1. Design and synthesis of shRNAs/ 4.2. Plants, virus maintenance and shRNAs infiltration / 4.3. Protein extraction and quantitation/ 4.4. Immunological-based assays for specific detection of CMV/ 4.5. RNA isolation and cDNA synthesis / 4.6. PCR, quantitative real-time PCR (qPCR) and primer efficiency test / 4.7. Statistical analysis/ 5. Conclusions/ References/ CAPÍTULO IV: TERCER ARTÍCULO / Aplicación tópica de dsRNAs, como alternativa al silenciamiento génico del virus del mosaico del pepino / RESUMEN / Abstract/ Introducción/ Materiales y métodos/ Extracción de RNA y síntesis de cDNA/ Diseño y síntesis de moléculas de silenciamiento de dsRNAs / dsRNAs/ Dot-blot / RT-qPCR. / Análisis estadísticos/ Resultados y discusión / Síntesis, calidad y rendimiento de dsRNAs/ Silenciamiento de CMV vía dsRNAs / Prueba serológica para detección de CMV/ Eficiencia de cebadores y especificidad/ RT-qPCR / Bibliografía/ CAPÍTULO V: DISCUSIÓN GENERAL / Bibliografía. | spa |
dc.format.mimetype | application/pdf | spa |
dc.language.iso | eng | spa |
dc.language.iso | spa | spa |
dc.title | Efectos de la aplicación de secuencias de ARN de cadena doble (ARNcd) en el silenciamiento del virus del mosaico del pepino (CMV) | spa |
dc.type | Trabajo de grado - Doctorado | spa |
dc.contributor.referee | Arboleda Valencia Jorge William | |
dc.contributor.referee | Betancur P. Jhon Fredy | |
dc.contributor.researchgroup | InGene (Categoría C) | spa |
dc.description.degreelevel | Doctorado | spa |
dc.description.notes | No autorizo publicación porque estamos pendientes de publicación de artículo científico | spa |
dc.identifier.instname | Universidad de Caldas | spa |
dc.identifier.reponame | Repositorio Universidad de Caldas | spa |
dc.identifier.repourl | https://repositorio.ucaldas.edu.co/mydspace | spa |
dc.publisher.faculty | Facultad de Ciencias Agropecuarias | spa |
dc.publisher.place | Manizales | spa |
dc.relation.references | Akbar, S.; Tahir, M.; Wang, M.B.; Liu, Q. Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant. BioMed Research International. 2017, 2017, 1-10; Doi: 10.1155/2017/1646140 | spa |
dc.relation.references | Aslam, U.; Tabassum, B.; Nasir, I.A.; Khan A.; Husnain, T. A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane. Transgenic Res. 2018, 27, 203–210; Doi: 10.1007/s11248-018-0066-1 | spa |
dc.relation.references | Bujarski, J.; Gallitelli, D.; García-Arenal, F.; Pallás, V.; Palukaitis, P.; Reddy, M.K.; Wang, A.; ICTV Report Consortium 2019. ICTV Virus Taxonomy Profile: Bromoviridae. Journal of General Virology 2019, 100, 1206–1207; Doi:10.1099/jgv.0.001282 | spa |
dc.relation.references | Csorba, T.; Kontra, L.; Burgyán, J. Viral silencing suppressors: tools forged to fine-tune host-pathogen coexistence. Virology. 2015, 479, 85–103; Doi: 10.1016/j.virol.2015.02.028 | spa |
dc.relation.references | Ding, S.W.; Anderson, B.J.; Haase, H.R.; Symons, R.H. New overlapping gene encoded by the cucumber mosaic virus genome. Virology 1994, 198, 593–601; Doi: 10.1006/viro.1994.1071 | spa |
dc.relation.references | Doolittle, S.P. A new infectious mosaic disease of cucumber. Phytopathology 1916, 6, 145–147. | spa |
dc.relation.references | Dubrovina, A.S.; Aleynova, O.A.; Kalachev, A.V.; Suprun, A.R.; Ogneva, Z.V.; Kiselev, K.V. Induction of Transgene Supression in Plants via External Application of Synthetic dsRNA. Int. J. Mol. Sci. 2019, 20, 1585; Doi: 10.3390/ijms20071585 | spa |
dc.relation.references | Florax, R.J.; Travisi, C.M.; Nijkamp, P.A. Meta-analysis of the willingness to pay for reductions in pesticide risk exposure. Eur. Rev. Agric. Econ. 2005, 32, 441–467; Doi: 10.1093/erae/jbi025 | spa |
dc.relation.references | García-Arenal, F. Palukaitis, P. Cucumber Mosaic Virus In: Encyclopedia of Virology, 3rd. ed.; Mahy, B. W. J., van Regenmortel, M. H. V., Eds.; Academic Press, Oxford, UK, 2008; pp. 614–619. doi: 10.1016/B978-012374410-4.00640-3 | spa |
dc.relation.references | Guo, J.; Gao, S.; Lin, Q.; Wang, H.; Que, Y.; Xu, L. Transgenic sugarcane resistant to Sorghum mosaic virus based on coat protein gene silencing by RNA interference. BioMed Research International 2015, 2015, 1-9; Doi: 10.1155/2015/861907 | spa |
dc.relation.references | Jacquemond, M. Cucumber mosaic virus. Adv Virus Res 2012, 84, 439-504; Doi:10.1016/B978-0-12-394314-9.00013-0. Kaldis, A.; Berbati, M.; Melita, O.; Reppa, C.; Holeva, M.; Otten, P.; Voloudakis, A. Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV. Mol. Plant Pathol. 2018, 19, 883–895; Doi: 10.1111/mpp.12572 | spa |
dc.relation.references | Kenyon, L.; Kumar, S., Tsai, W.S.; Hughes, J.D.A. Chapter Six - Virus diseases of peppers (Capsicum spp.) and their control. In Advances in virus research; Loebenstein, G., Katis, N., Eds.; Academic Press: Waltham, USA, 2014; Volume 90, pp. 297-354; Doi: https://doi.org/10.1016/B978-0-12-801246-8.00006-8. | spa |
dc.relation.references | Kim, N.Y.; Baek, J.Y.; Choi, H.S.; Chung, I.S.; Shin, S.; Lee, J.I.; Yang. J.M. Short-hairpin RNA-mediated gene expression interference in Trichoplusia ni cells. J. Microbiol. Biotechnol. 2012, 22, 190-198; Doi: 10.4014/jmb.1108.08045 | spa |
dc.relation.references | Konakalla, N.C.; Kaldis, A.; Berbati, M.; Masarapu, H.; Voloudakis, A.E. Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco. Planta. 2016, 244, 961–969; Doi: 10.1007/s00425-016-2567-6 | spa |
dc.relation.references | Lau, S.E.; Mazumdar, P.; Hee, T.W.; Song, A.L.A.; Othman, R.Y.; Harikrishna, J.A. Crude extracts of bacterially-expressed dsRNA protect orchid plants against Cymbidium mosaic virus during transplantation from in vitro culture. The Journal of Horticultural Science and Biotechnology 2014, 89, 569-576; Doi: 10.1080/14620316.2014.11513122 | spa |
dc.relation.references | Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001, 25, 402–408; Doi: 10.1006/meth.2001.1262 | spa |
dc.relation.references | Mitter N.; Worrall, E.A.; Robinson, K.E.; Li, P.; Jain, R.G.; Taochy, C.; Fletcher, S.J.; Carroll, B.J.; Lu, G.Q.; Xu, Z.P. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature plants 2017 3, 16207; Doi: 10.1038/nplants.2016.207 | spa |
dc.relation.references | Namgial, T.; Kaldis, A.; Chakraborty, S.; Voloudakis, A. Topical application of double-stranded RNA molecules containing sequences of Tomato leaf curl virus and Cucumber mosaic virus confers protection against the cognate viruses. Physiological and Molecular Plant Pathology 2019, 108, 101432; Doi: 10.1016/j.pmpp.2019.101432. | spa |
dc.relation.references | Rahman, M.S.; Ahmed, A.U.; Jahan, K.; Khatun, F. Management of Cucumber Mosaic Virus (CMV) infecting cucumber in bangladesh. Bangladesh J. Agril. Res. 2020, 45, 65-76. | spa |
dc.relation.references | Scholthof, K.B.; Adkins, S.; Czosnek, H.; Palukaitis, P.; Jacquot, E.; Hohn, T.; Hohn, B.; Saunders, K,; Candresse, T.; Ahlquist, P.; Hemenway, C.; Foster, G.D. (2011) Top 10 plant viruses in molecular plant pathology. Molecular plant pathology 2011, 12, 938–954; Doi: https://doi.org/10.1111/j.1364-3703.2011.00752.x | spa |
dc.relation.references | Schwind, N.; Zwiebel, M.; Itaya, A.; Ding, B.; Wang, M.B.; Krczal, G.; Wassenegger, M. RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct. Mol. Plant. Pathol. 2009, 10, 459–469; Doi: 10.1111/j.1364-3703.2009.00546.x. | spa |
dc.relation.references | Sharma, V.K.; Sanghera, G.S.; Kashyap, P.L.; Sharma, B.B.; Chandel, C. RNA interference: A novel tool for plant disease management. African Journal of Biotechnology 2013, 12, 2303-2312; Doi: 10.5897/AJB12.2791 | spa |
dc.relation.references | Sharp, P.A. RNA interference—2001. Genes Dev. 2001, 15, 485–490; Doi:10.1101/gad.880001 | spa |
dc.relation.references | Shen, W.; Yang, G.; Chen, Y.; Yan, P.; Tuo, D.; Li, X.; Zhou, P. Resistance of non-transgenic papaya plants to papaya ringspot virus (PRSV) mediated by intron-containing hairpin dsRNAs expressed in bacteria. Acta Virol. 2014, 58, 261–266; Doi: 10.4149/av_2014_03_261 | spa |
dc.relation.references | Tabassum, B.; Nasir, I.A.; Khan, A.; Aslam, U.; Tariq, M.; Shahid, N.; Husnain, T. Short hairpin RNA engineering: In planta gene silencing of potato virus Y. Crop Protection. 2016, 86, 1-8; Doi: 10.1016/j.cropro.2016.04.005 | spa |
dc.relation.references | Vadlamudi, T.; Patil, B.L.; Kaldis, A.; Gopal, D.V.; Mishra, R.; Berbati, M.; Voloudakis, A. DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya. J. virol. methods 2020, 275, 113750; Doi: 10.1016/j.jviromet.2019.113750 | spa |
dc.relation.references | Voinnet, O. Induction and suppression of RNA silencing: insights from viral infections. Nature Reviews Genetics 2005, 6, 206–220; Doi: 10.1038/nrg1555 | spa |
dc.relation.references | Wang, F.; Li, W.; Zhu, J.; Fan, F.; Wang, J.; Zhong, W.; Wang, M.B.; Liu, Q.; Zhu, Q.H.; Zhou, T.; Lan, Y.; Zhou, Y.; Yang, J. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf. Virus Int. J. Mol. Sci. 2016, 17, 705; Doi: 10.3390/ijms17050705 | spa |
dc.relation.references | Wang, M.B.; Metzlaff, M. RNA silencing and antiviral defense in plants. Curr. Opin. Plant. Biol. 2005, 8, 216–222; Doi: 10.1016/j.pbi.2005.01.006 | spa |
dc.relation.references | Watters, K.E.; Choudhary, K.; Aviran, S.; Lucks, J.B.; Perry, K.L.; Thompson, J.R. Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements. Nucleic Acids Research 2018 46, 2573–2584; Doi: https://doi.org/10.1093/nar/gkx1273 | spa |
dc.relation.references | Worrall, E.A.; Bravo-Cazar, A.; Nilon, A.T.; Fletcher, S.J.; Robinson, K.E.; Carr, J.P.; Mitter, N. Exogenous application of RNAi-inducing double-stranded RNA inhibits aphid-mediated tansmission of a plant virus. Front. Plant Sci. 2019, 10, 265; Doi: 10.3389/fpls.2019.00265. | spa |
dc.relation.references | Zhang, H.; Demirer, G.S.; Zhang, H.; Ye, T.; Goh, N.S.; Aditham, A.J.; Cunningham, F.J.; Fan, C.; Landry, M.P. DNA nanostructures coordinate gene silencing in mature plants. Proc. Natl. Acad. Sci. 2019, 116, 7543-7548; Doi: 10.1073/pnas.1818290116 | spa |
dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.subject.lemb | Genética | |
dc.subject.lemb | Genes | |
dc.subject.proposal | CMV | spa |
dc.subject.proposal | Proteina de la cápside | spa |
dc.subject.proposal | Nicotiana benthamiana | spa |
dc.subject.proposal | Silenciamiento génico | spa |
dc.subject.proposal | shRNA | eng |
dc.subject.proposal | dsRNA | eng |
dc.type.coar | http://purl.org/coar/resource_type/c_db06 | spa |
dc.type.content | Text | spa |
dc.type.driver | info:eu-repo/semantics/doctoralThesis | spa |
dc.type.version | info:eu-repo/semantics/publishedVersion | spa |
oaire.version | http://purl.org/coar/version/c_ab4af688f83e57aa | spa |
oaire.accessrights | http://purl.org/coar/access_right/c_14cb | spa |
dc.description.degreename | Doctor(a) en Ciencias Agrarias | spa |
dc.publisher.program | Doctorado en Ciencias Agrarias | spa |
dc.description.researchgroup | Evaluación y validación del silenciamiento génico como estrategia para el manejo de fitopatógenos | spa |
dc.rights.coar | http://purl.org/coar/access_right/c_14cb | spa |