RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico
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| Autor principal: | |
|---|---|
| Data de publicació: | 2016 |
| Format: | Master thesis |
| Idioma: | por |
| Font: | Repositório Institucional da UPF |
| Download full: | https://repositorio.upf.br/handle/123456789/9235 |
Sumari: | Helicoverpa armigera is a pest insect of several economically important crops. Research groups have studied RNA interference (RNAi) to knock down expression of vital genes within target pests. Successful reduction of the expression of these genes can lead to mortality and affect the insect development. The objective of this work was to evaluate the ability of orally introduced double strand RNA (dsRNA) matching the H. armigera larvae rieske gene to induce an RNAi response and to cause mortality. The rieske gene encodes a mitochondrial protein involved in the electron transport and energy production. We hypothesize that dsRNA will enter the gut epithelial cells and induce an RNAi response that reduces rieske gene expression, and consequently, reduce energy production. This should reduce the cells ability to perform digestive functions resulting in larvae mortality. The experiments were performed in the Plant Biotechnology and Entomology Laboratories of the University of Passo Fundo. Initially, a profile of developmental changes in the rieske transcript was developed for dissected digestive tracts and whole insects and appropriated reference genes were identified for use with RT-qPCR. Second, the dsRNA dose most effective at inducing larvae mortality was determined. Subsequently, the occurrence of gene silencing and its relationship with larval mortality and development was evaluated. The RT-qPCR reference genes found to be the most stable for normalization of rieske gene expression were elongation factor 1-α (ef1-α) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The rieske dsRNA dose of 0,3 µg.cm-2was most effective at inducing larvae mortality, reducing weight gain and inhibiting leaf consumption. Delivering 0.3 µg.cm-2of dsRNA rieske every two days to soybean leaf discs caused a reduction in the rieske gene transcripts and increased Helicoverpa armigera larval mortality. But when the feeding of leaf discs treated with dsRNA was interrupted with untreated leaf disks similar effects were not observed. These results indicate that the RNAi mediated gene silencing of rieske has the potential to be used as H. armigera control, however for increased effectiveness, the delivery of the dsRNA must be continuous. |
| _version_ | 1871607643627323392 |
|---|---|
| author | Ceccon, Cássia Canzi |
| author_browse | Ceccon, Cássia Canzi |
| author_facet | Ceccon, Cássia Canzi |
| author_role | author |
| collection | Repositório Institucional da UPF |
| dc.contributor.author.fl_str_mv | Ceccon, Cássia Canzi |
| dc.contributor.none.fl_str_mv | Grando, Magali Ferrari http://lattes.cnpq.br/7254787776404840 |
| dc.date.none.fl_str_mv | 2016-04-29 2025-06-20T16:41:42Z |
| dc.format.none.fl_str_mv | application/pdf application/pdf |
| dc.identifier.uri.fl_str_mv | CECCON, Cássia Canzi. RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico. 2016. 150 f. Dissertação (Mestrado em Agronomia) - Universidade de Passo Fundo, Passo Fundo, RS, 2016. https://repositorio.upf.br/handle/123456789/9235 |
| dc.language.iso.fl_str_mv | por |
| dc.publisher.none.fl_str_mv | Universidade de Passo Fundo Faculdade de Agronomia e Medicina Veterinária – FAMV Brasil UPF Programa de Pós-Graduação em Agronomia |
| dc.rights.driver.fl_str_mv | info:eu-repo/semantics/openAccess |
| dc.source.none.fl_str_mv | reponame:Repositório Institucional da UPF instname:Universidade de Passo Fundo (UPF) instacron:UPF |
| dc.subject.por.fl_str_mv | Lagarta Sequência de bases Biotecnologia agrícola Pragas agrícolas - Controle CIENCIAS AGRARIAS::AGRONOMIA |
| dc.title.none.fl_str_mv | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico RNA interference as an alternative for the control of helicoverpa armigera through gene silencing |
| dc.type.driver.fl_str_mv | info:eu-repo/semantics/masterThesis |
| dc.type.status.fl_str_mv | info:eu-repo/semantics/publishedVersion |
| description | Helicoverpa armigera is a pest insect of several economically important crops. Research groups have studied RNA interference (RNAi) to knock down expression of vital genes within target pests. Successful reduction of the expression of these genes can lead to mortality and affect the insect development. The objective of this work was to evaluate the ability of orally introduced double strand RNA (dsRNA) matching the H. armigera larvae rieske gene to induce an RNAi response and to cause mortality. The rieske gene encodes a mitochondrial protein involved in the electron transport and energy production. We hypothesize that dsRNA will enter the gut epithelial cells and induce an RNAi response that reduces rieske gene expression, and consequently, reduce energy production. This should reduce the cells ability to perform digestive functions resulting in larvae mortality. The experiments were performed in the Plant Biotechnology and Entomology Laboratories of the University of Passo Fundo. Initially, a profile of developmental changes in the rieske transcript was developed for dissected digestive tracts and whole insects and appropriated reference genes were identified for use with RT-qPCR. Second, the dsRNA dose most effective at inducing larvae mortality was determined. Subsequently, the occurrence of gene silencing and its relationship with larval mortality and development was evaluated. The RT-qPCR reference genes found to be the most stable for normalization of rieske gene expression were elongation factor 1-α (ef1-α) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The rieske dsRNA dose of 0,3 µg.cm-2was most effective at inducing larvae mortality, reducing weight gain and inhibiting leaf consumption. Delivering 0.3 µg.cm-2of dsRNA rieske every two days to soybean leaf discs caused a reduction in the rieske gene transcripts and increased Helicoverpa armigera larval mortality. But when the feeding of leaf discs treated with dsRNA was interrupted with untreated leaf disks similar effects were not observed. These results indicate that the RNAi mediated gene silencing of rieske has the potential to be used as H. armigera control, however for increased effectiveness, the delivery of the dsRNA must be continuous. |
| eu_rights_str_mv | openAccess |
| format | masterThesis |
| id | UPF_cfd4dfc6c56dab6c092c1332cd14fb96 |
| identifier_str_mv | CECCON, Cássia Canzi. RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico. 2016. 150 f. Dissertação (Mestrado em Agronomia) - Universidade de Passo Fundo, Passo Fundo, RS, 2016. |
| instacron_str | UPF |
| institution | UPF |
| instname_str | Universidade de Passo Fundo (UPF) |
| language | por |
| network_acronym_str | UPF |
| network_name_str | Repositório Institucional da UPF |
| oai_identifier_str | oai:repositorio.upf.br:123456789/9235 |
| publishDate | 2016 |
| publishDateSort | 2016 |
| publisher.none.fl_str_mv | Universidade de Passo Fundo Faculdade de Agronomia e Medicina Veterinária – FAMV Brasil UPF Programa de Pós-Graduação em Agronomia |
| reponame_str | Repositório Institucional da UPF |
| repository.mail.fl_str_mv | jucelei@upf.br||biblio@upf.br |
| repository.name.fl_str_mv | Repositório Institucional da UPF - Universidade de Passo Fundo (UPF) |
| repository_id_str | 1610 |
| spelling | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênicoRNA interference as an alternative for the control of helicoverpa armigera through gene silencingLagartaSequência de basesBiotecnologia agrícolaPragas agrícolas - ControleCIENCIAS AGRARIAS::AGRONOMIAHelicoverpa armigera is a pest insect of several economically important crops. Research groups have studied RNA interference (RNAi) to knock down expression of vital genes within target pests. Successful reduction of the expression of these genes can lead to mortality and affect the insect development. The objective of this work was to evaluate the ability of orally introduced double strand RNA (dsRNA) matching the H. armigera larvae rieske gene to induce an RNAi response and to cause mortality. The rieske gene encodes a mitochondrial protein involved in the electron transport and energy production. We hypothesize that dsRNA will enter the gut epithelial cells and induce an RNAi response that reduces rieske gene expression, and consequently, reduce energy production. This should reduce the cells ability to perform digestive functions resulting in larvae mortality. The experiments were performed in the Plant Biotechnology and Entomology Laboratories of the University of Passo Fundo. Initially, a profile of developmental changes in the rieske transcript was developed for dissected digestive tracts and whole insects and appropriated reference genes were identified for use with RT-qPCR. Second, the dsRNA dose most effective at inducing larvae mortality was determined. Subsequently, the occurrence of gene silencing and its relationship with larval mortality and development was evaluated. The RT-qPCR reference genes found to be the most stable for normalization of rieske gene expression were elongation factor 1-α (ef1-α) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The rieske dsRNA dose of 0,3 µg.cm-2was most effective at inducing larvae mortality, reducing weight gain and inhibiting leaf consumption. Delivering 0.3 µg.cm-2of dsRNA rieske every two days to soybean leaf discs caused a reduction in the rieske gene transcripts and increased Helicoverpa armigera larval mortality. But when the feeding of leaf discs treated with dsRNA was interrupted with untreated leaf disks similar effects were not observed. These results indicate that the RNAi mediated gene silencing of rieske has the potential to be used as H. armigera control, however for increased effectiveness, the delivery of the dsRNA must be continuous.A lagarta Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) é considerada inseto-praga de culturas de importância econômica. Grupos de pesquisas têm estudado o RNA de interferência (RNAi) para silenciamento de genes vitais do inseto alvo. A redução da expressão desses genes pode causar mortalidade e afetar o desenvolvimento do inseto alvo. O objetivo deste trabalho foi avaliar a habilidade do RNAdf com complementariedade à porção do gene rieske de H. armigera em induzir a resposta de RNAi e causar mortalidade, quando disponibilizado oralmente. O gene rieske codifica para uma proteína mitocondrial envolvida no transporte de elétrons e produção de energia. A hipótese testada foi a de que o RNAdf entra nas células epiteliais do intestino induzindo a resposta de RNAi, reduzindo a expressão do gene rieske e consequentemente, a produção de energia. Este efeito reduziria a habilidade das células em realizar as funções digestivas resultando na mortalidade das larvas. Os experimentos foram realizados nos Laboratórios de Biotecnologia Vegetal e de Entomologia da Universidade de Passo Fundo. Inicialmente, foi determinado o perfil de expressão do gene rieske durante o desenvolvimento larval no tecido digestório e do restante do corpo e foram identificados os genes de referência apropriados para uso em RT-qPCR. Em seguida, foi determinada a dose de RNAdf mais efetiva na indução da mortalidade das larvas. Posteriormente, foi avaliada a ocorrência do silenciamento gênico e a sua relação com a mortalidade e o desenvolvimento das larvas. Os genes de referência mais estáveis para normalização da expressão do gene rieske por RT-qPCR foram fator de elongação 1-¿ (ef1-¿) e gliceraldeído-3-fosfato desidrogenase (GAPDH). A dose de 0,3 ¿g.cm-2 de RNAdf rieske foi mais efetiva na indução da mortalidade, redução do ganho de peso e consumo. A disponibilização, a cada dois dias, de discos foliares de soja contendo 0,3 ¿g.cm-2 de RNAdf rieske causou a redução dos transcritos do gene rieske e mortalidade das larvas de H. armigera. Porém, quando a alimentação com discos foliares tratados com RNAdf foi intercalada com discos foliares não tratados os efeitos observados não foram similares. Esses resultados indicam que o silenciamento do gene rieske mediado por RNAi tem potencial para ser utilizado para controle de H. armigera, porém, para maior efetividade, a disponibilização do RNAdf deve ser contínua.Universidade de Passo FundoFaculdade de Agronomia e Medicina Veterinária – FAMVBrasilUPFPrograma de Pós-Graduação em AgronomiaGrando, Magali Ferrarihttp://lattes.cnpq.br/7254787776404840Ceccon, Cássia Canzi2025-06-20T16:41:42Z2016-04-29info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfapplication/pdfCECCON, Cássia Canzi. RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico. 2016. 150 f. Dissertação (Mestrado em Agronomia) - Universidade de Passo Fundo, Passo Fundo, RS, 2016.https://repositorio.upf.br/handle/123456789/9235porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UPFinstname:Universidade de Passo Fundo (UPF)instacron:UPF2025-12-30T19:50:46Zoai:repositorio.upf.br:123456789/9235Repositório InstitucionalPRIhttp://repositorio.upf.br/oai/requestjucelei@upf.br||biblio@upf.bropendoar:16102025-12-30T19:50:46Repositório Institucional da UPF - Universidade de Passo Fundo (UPF) |
| spellingShingle | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico Ceccon, Cássia Canzi Lagarta Sequência de bases Biotecnologia agrícola Pragas agrícolas - Controle CIENCIAS AGRARIAS::AGRONOMIA |
| status_str | publishedVersion |
| title | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico |
| title_full | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico |
| title_fullStr | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico |
| title_full_unstemmed | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico |
| title_short | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico |
| title_sort | RNA interferência como alternativa para o controle de helicoverpa armigera mediante silenciamento gênico |
| topic | Lagarta Sequência de bases Biotecnologia agrícola Pragas agrícolas - Controle CIENCIAS AGRARIAS::AGRONOMIA |
| url | https://repositorio.upf.br/handle/123456789/9235 |