Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O feijÃo-de-corda [Vigna unguiculata (L.) Walp.] tem grande importÃncia socioeconÃmica no Nordeste brasileiro. Entretanto, sua produÃÃo à baixa devida a diversos fatores abiÃticos e biÃticos. Dentre os fatÃres biÃticos, o vÃrus do mosaico severo do caupi (CPSMV, famÃlia Secoviridae) apresenta grande destaque, por causar a virose que mais acomete essa cultura no paÃs. Embora existam cultivares resistentes ao vÃrus, nÃo se sabe quais os mecanismos de defesa envolvidos. Por essa razÃo, foi elaborado um estudo comparativo entre cultivares resistentes e susceptÃveis, utilizando duas abordagens experimentais. Na abordagem bioquÃmica, possÃveis diferenÃas de atividades de enzimas relacionadas ao estresse oxidativo (dismutase do superÃxido, peroxidase do ascorbato, peroxidase) e à patogÃnese (β-1,3-glucanase e quitinase), alÃm da fenilamÃnia liase, e teores de H2O2 foram estudadas nos cultivares PitiÃba (susceptÃvel) e Macaibo (resistente). ApÃs tratamento com o CPSMV ou com apenas carborundum (plantas controles), foram realizadas coletas nos tempos de 6, 12, 24, 48 e 72 h, tendo sido realizadas as anÃlises bioquÃmicas nos extratos protÃicos obtidos das folhas secundÃrias. Foi verificado que, de maneira geral, a resposta entre os cultivares nÃo diferiram significamente, sugerindo que os mecanismos de defesa de feijÃo-de-corda sejam diferentes da resposta clÃssica de defesa. Na segunda abordagem, molecular, as sequÃncias nucleotÃdicas dos genes codificantes para os fatores de iniciaÃÃo de traduÃÃo (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G e nCBP) e as sequÃncias primÃrias putativas das proteÃnas correspondentes foram analisados, no intuito de se averiguar a existÃncia de padrÃes de polimorfismos entre cultivares resistentes e susceptÃveis, que pudessem estar relacionados à defesa constitutiva conferida por genes recessivos. ApÃs anÃlise das sequÃncias, foi observado que eIF4E apresentava polimorfismos entre os cultivares, sendo que, em pelo menos duas posiÃÃes nas sequÃncias primÃrias putativas do fator (68 e 108), existiram diferenÃas entre cultivares susceptÃveis e resistentes (Arg68/Pro68; Val108 ou Pro108/Ala108). A modelagem molecular revelou que as diferenÃas em aminoÃcidos situam-se em dois loops externos, prÃximos ao domÃnio de ligaÃÃo ao capacete (m7G), bastante relatados em casos de resistÃncia recessiva para a famÃlia Potyviridae. AtravÃs de estudos de imunodectecÃÃo posterior ao passo cromatogrÃfico em coluna de afinidade, foi observado que as mudanÃas de aminoÃcidos nÃo comprometiam, a capacidade de eIF4E em se ligar ao m7G in vitro. Entretanto, como foram observadas duas variantes para eIF4E, entre cultivares resistentes e susceptÃveis ao CPSMV, em regiÃes prÃximas espacialmente, nÃo se pode descartar a hipÃtese de que a resistÃncia recessiva constitutiva esteja associada com essas mutaÃÃes detectadas nessas sequÃncias, que iriam modificar, consequentemente, a interaÃÃo da VPg viral com eIF4E in vivo. / Cowpea [(Vigna unguiculata (L.) Walp.)] has a major socioeconomic importance in Northeastern Brazil. However, its production is low due abiotic and biotic factors. Amongst the biotic factors the cowpea severe mosaic virus (CPSMV, Secoviridae family) has a great importance because it causes the most prevalent and serious virus disease that affects this crop in the country. Although there are resistant cultivars to CPSMV, the defense mechanisms involved is not understood. For this reason, a comparative study was conducted between resistant and susceptible cultivars, using two experimental approaches. In the first one, the biochemical approach, possible differences of enzyme activities related to oxidative stress (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase) and pathogenesis (β-1,3-glucanase and chitinase), in addition of phenyl amonium lyase, and H2O2 generation in the leaves of the cultivars PitÃuba (susceptible) and Macaibo (resistant) were analyzed. The secondary leaves were harvested at 6, 12, 24, 48, 72 h after treatment with CPSMV or carborundum (controls) and these above parameters were measures in the protein extracts obtained. It was shown that, in general, the response amongst the cultivars did not differ significantly, suggesting that the defense mechanisms of cowpea are different from the classic response of defense observed for several plant species. In the second approach, molecular, the nucleotide sequences of the genes that code for the translation initiation factors (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G and nCBP) and the primary strucuture of the correspondent putative proteins were analyzed in order to search patterns of polymorphis between the studied cowpea cultivars that could be related to a constitutive defense conferred by recessive genes. After sequence analysis, it was found that eIF4E showed polymorphisms between cultivars, and, in at least two positions (68 and 108), there were differences between susceptible and resistant cultivars (Arg68/Pro68; Val108 or Pro108/Ala108). The molecular modeling revealed that differences in amino acid are located in two external loops close to the cap (m7G) binding domain, well reported in cases of recessive resistance within the Potyviridae family. Through immunodetection studies with the leaf extracts and the protein fractions obtained after the affinity chromatography on a Sepharose-7-metil-guanosina column, it was found that the amino acid mutations found did not impair the ability of eIF4E to bind to M7G in vitro. However, as it was observed two variants for eIF4E comparing the resistant and susceptible cultivars to CPSMV, at spatially neighboring regions, it could not be ruled out the hypothesis that this constitutive/recessive resistant trait is correlated with these mutations detected, which could impair, consequently, the in vivo interaction of eIF4E with the viral VPg.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:4599 |
| Date | 12 August 2011 |
| Creators | Vladimir GonÃalves MagalhÃes |
| Contributors | Josà Tadeu Abreu de Oliveira, Thalles Barbosa Grangeiro, Josà Evando Aguiar Beserra JÃnior |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em BioquÃmica, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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