Determinación in vitro e in vivo de las propiedades insecticidas de las lectinas P2 y P4 de semillas de Galactia lindenii sobre un lepidóptero

dc.contributor.advisorReyes Montaño, Edgar Antoniospa
dc.contributor.authorCasas Corredor, Zulma Yaniraspa
dc.contributor.researchgroupGrupo de Investigación en Proteinas Gripspa
dc.contributor.subjectmatterexpertVega, Nohoraspa
dc.date.accessioned2025-09-22T20:08:20Z
dc.date.available2025-09-22T20:08:20Z
dc.date.issued2023-03-15
dc.descriptionilustraciones, diagramas, fotografíasspa
dc.description.abstractAlgunas lectinas de origen vegetal se caracterizan por su efecto entomotóxico, provocando efectos antinutritivos, inhibición del crecimiento, disminución en el desarrollo y aumento en la mortalidad de los insectos, cuando se administran por vía oral en dieta artificial o se expresan en plantas transgénicas. En este trabajo se estudió la actividad bioinsecticida de las lectinas de Galactia lindenii, LGL-P2 y LGL-P4, la lectina LGL-P2 es específicas por trisacárido H-tipo II (Fucα1,2Galβ1,4GlcNAc), mientras que la lectina LGL-P4 específica por glucosa (Glc)/ Manosa (Man). Las lectinas fueron aisladas a partir de las semillas de Galactia lindenii y purificadas por diferentes técnicas cromatográficas, como intercambio aniónico, afinidad y filtración en gel. Las lectinas se obtuvieron en diferentes cantidades, 6% (LGL-P2) y 0.012 % (LGL-P4); por SDS PAGE mostraron bandas de ~25 kDa y ~ 30 kDa para los monómeros de LGL-P2 y LGL-P4 respectivamente. Se llevó a cabo la caracterización estructural de la LGL-P4, mediante digestión tríptica y secuenciación de péptidos por espectrometría de masas en tándem, alcanzado una secuencia parcial con 65,8 % de cobertura; por predicción computacional se determinó que pertenece a las lectinas tipo Leguminosa (Tipo L) y que presenta una alta posibilidad de interacción con ácido-N-acetilneuramínico (NeuAc), carbohidrato presente en el intestino de insectos. Por otra parte, las lectinas mostraron un efecto dependiente de la concentración sobre la línea celular de insecto CF203, dado que disminuyeron la viabilidad celular con 10 μM (LGL-P2) y 3 μM (LGL-P4); contrariamente, con 0,03 μM LGL-P4 promovió su proliferación. Para complementar los estudios de actividad biológica, se determinó el efecto bioinsecticida de la lectina LGL-P2, realizando ensayos in vivo con larvas de primer estadio de Spodoptera frugiperda alimentadas con dieta artificial que incluyó diferentes cantidades de la lectina, observándose hasta un 66 % de mortalidad en el día once. Se determinó que LGL-P2 y LGL-P4 se unen en el intestino medio de larvas de cuarto estadio de S. frugiperda alterando la membrana peritrófica y células del intestino medio. Los resultados mostraron que las lectinas de Galactia lindenii tienen un gran potencial para el manejo integrado de insectos como S. frugiperda y por lo tanto se podrían emplear como agentes biológicos para controlar los insectos plaga, que afectan los cultivos de gran interés económico. (Texto tomado de la fuente).spa
dc.description.abstractSome vegetal lectins have an entomotoxic effect, which causes (i) antinutritive effects, (Tereshchenkova et al.) growth inhibition, (iii) development decline, and (iv) mortality increase in insects, when administered orally in artificial diet or expressed in transgenic plants. This work studied the bioinsecticide activity of lectins found in Galactia lindenii, i.e., LGL-P2, and LGL-P4. Lectin LGL-P2 is specific for the type II H- trisaccharide (Fucα1,2Galβ1,4GlcNAc), while lectin LGL-P4 is specific for glucose (Glc)/Mannose (Man). These lectins were isolated from Galactia lindenii seeds and purified with different chromatographic technics, such as: anionic exchange, affinity, and gel filtration. The lectins were obtained in different amounts, 6 % (LGL-P2) and 0.012 % (LGL-P4). Analysis of the lectins by SDS PAGE indicated bands of ∼25 kDa and ∼30 kDa for the monomers of LGL-P2 y LGL-P4, respectively. The structural characterization of LGL-P4 was performed through tryptic digestion and peptide sequencing by tandem mass spectrometry, reaching a partial sequence of 65,8 % coverage. Based on the computational prediction, it was identified that the LGL-P4 lectin was a legume type (Type L), with a high probability for interaction with acid-N-acetylneuraminic (NeuAc); this carbohydrate is present in the intestine of insects. In addition, the lectins showed a concentration effect on the insect cell line CF203, as they decreased the cell viability at 10 μM (LGL-P2) and 3 μM (LGL-P4); but promoted proliferation at 0,03 μM (LGL-P4). The bioinsecticide effect of LGL-P2 lectin was evaluated by in vivo trials in which Spodoptera frugiperda larvae in the first instar were fed. The artificial diet used in the trials included different lectin concentrations. Up to a 66% mortality at the eleventh day was found. Furthermore, the studies showed that LGL-P2 and LGL-P4 bond to the midgut at the fourth instar of S. frugiperda, altering the peritrophic membrane and the midgut cells. Results showed that Galactia lindenii lectins have excellent potential for integrated insect management of S. frugiperda. Therefore, they could be used as biological agents to control pest insects, which affect crops of great economic interest.eng
dc.description.degreelevelDoctoradospa
dc.description.degreenameDoctor en Biotecnologíaspa
dc.description.methodsSe siguió la metodología descrita por (Almanza et al., 2004). Se recolectaron semillas de Galactia lindenii en inmediaciones de la laguna de Fúquene, se dejaron testigos en el instituto de Ciencias Naturales (ICN) y se registraron con voucher COL 15115 y 580116. Para estudiar esta especie nativa se utilizó el contrato de acceso a recurso genético otorgado por el Ministerio de Ambiente y Desarrollo Sostenible RGE 246. Las semillas se molieron hasta obtener una harina y para obtener las proteínas solubles se utilizaron 5 o 10 g de harina y se adicionaron 50 mL o 100 mL de buffer acetato-acético, tioúrea pH 5,0 (100mM de acetato, 150mM NaCl y 5 mM tioúrea) (1:10 p/v), o con buffer fosfato salino (PBS 1X) pH 7.2 (Na2HPO4 20 mM), con tiourea 5 mM, la extracción se dejó en agitación continua durante 8-12 horas a 4 °C. Posteriormente se centrifugó a 12.000 r.p.m durante 45 min a 4 °C, se separó el pellet y con el mismo volumen de buffer se realizó el procedimiento dos veces más. Se obtuvieron tres extractos y se unieron para obtener el extracto total (ET). El extracto total obtenido fue concentrado en una celda con agitación para ultrafiltración (Amicon) usando una membrana de celulosa con límite de tamaño molecular (MW cut off) de 10 kDa y almacenado a -20 °C hasta posteriores ensayos. Se utilizó una columna de 12 cm x 2 cm con DEAE-Sephadex (matriz de dextrán con grupo dietilaminoetil) a la que se le adicionó extracto total. La columna se equilibró con PBS 1 X; en cada corrida la fracción no retenida (DEAE-FNR), se eluyó con buffer PBS 1X (Na2HPO4 20mM, NaH2PO4 20 mM, NaCl 150 mM) pH 7.2-7.4, y la fracción retenida (DEAE-FR), se eluyó aumentando la fuerza iónica del PBS 1X con NaCl 0,05M (Figura 2-1) El seguimiento de las proteínas se realizó midiendo absorbancias a 220 y 280 nm. Las dos fracciones se concentraron en una celda de ultrafiltración (Amicon) usando una membrana de celulosa límite de tamaño molecular (MW cut off) de 10kDa; posteriormente se almacenaron a -20 °C hasta su uso en los ensayos posteriores. Para la preparación del soporte se siguió la metodología descrita por (Hermanson et al., 1992) (Anexo-A), se utilizó una columna de 30 X 1.2 cm y se adicionaron 15mL de la fracción no retenida de la cromatografía de intercambio iónico (DEAE-FNR). La columna se equilibró previamente con PBS 1X (Na2HPO4 20mM, NaH2PO4 20 mM, NaCl 150 mM) pH 7,2-7,4. La fracción no retenida (FNR-Lac) se eluyó utilizando PBS 1X, mientras que la fracción retenida (FR-Lac) se eluyó con PBS 1X Lac 0.2 M. Se realizó el seguimiento con absorbancia de 220 y 280 nm. La fracción retenida se dializó contra una solución de bicarbonato de amonio 20 mM (NH4HCO3) (Figura 2-1), realizando tres cambios, se liofilizó y almacenó para posteriores ensayos.spa
dc.description.researchareaLectinas de leguminosasspa
dc.description.sponsorshipColcienciasspa
dc.format.extentxii, 198 páginasspa
dc.format.mimetypeapplication/pdf
dc.identifier.instnameUniversidad Nacional de Colombiaspa
dc.identifier.reponameRepositorio Institucional Universidad Nacional de Colombiaspa
dc.identifier.repourlhttps://repositorio.unal.edu.cospa
dc.identifier.urihttps://repositorio.unal.edu.co/handle/unal/88933
dc.language.isospa
dc.publisherUniversidad Nacional de Colombiaspa
dc.publisher.branchUniversidad Nacional de Colombia - Sede Bogotáspa
dc.publisher.facultyFacultad de Cienciasspa
dc.publisher.placeBogotá, Colombiaspa
dc.publisher.programBogotá - Ciencias - Doctorado en Biotecnologíaspa
dc.relation.indexedAgrosaviaspa
dc.relation.indexedAgrovocspa
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dc.rights.accessrightsinfo:eu-repo/semantics/openAccess
dc.rights.licenseAtribución-NoComercial-SinDerivadas 4.0 Internacional
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.agrovocPropiedades plaguicidasspa
dc.subject.agrovocPesticidal propertieseng
dc.subject.agrovocPéptidosspa
dc.subject.agrovocPeptideseng
dc.subject.ddc570 - Biología::572 - Bioquímicaspa
dc.subject.ddc590 - Animales::595 - Artrópodosspa
dc.subject.proposalLeguminosasspa
dc.subject.proposalGalactia lindeniispa
dc.subject.proposalFabáceaspa
dc.subject.proposalLectinasspa
dc.subject.proposalInsecticidaspa
dc.subject.proposalInsectistáticospa
dc.subject.proposalLegumensspa
dc.subject.proposalLectinseng
dc.subject.proposalGalactia lindeniieng
dc.subject.proposalInsecticideeng
dc.subject.proposalInsectistaticeng
dc.subject.wikidataLepidopteraspa
dc.subject.wikidataLepidopteraeng
dc.subject.wikidatacarbohydrate binding proteinseng
dc.titleDeterminación in vitro e in vivo de las propiedades insecticidas de las lectinas P2 y P4 de semillas de Galactia lindenii sobre un lepidópterospa
dc.title.translatedIn vitro and in vivo determination of the insecticidal properties of P2 and P4 lectins from Galactia lindenii seeds against a lepidopteraneng
dc.typeTrabajo de grado - Doctoradospa
dc.type.coarhttp://purl.org/coar/resource_type/c_db06
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dc.type.contentText
dc.type.driverinfo:eu-repo/semantics/doctoralThesis
dc.type.redcolhttp://purl.org/redcol/resource_type/TD
dc.type.versioninfo:eu-repo/semantics/acceptedVersion
dcterms.audience.professionaldevelopmentInvestigadoresspa
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Tesis de Doctorado en Biotecnología

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