Análise proteômica da embriogênese somática e da aquisição de competência embriogência de Ocotea catharinensis Mez (Lauraceae)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Ocotea catharinensis is a forest tree species from Mata Atlântica that has economic importance due to its wood and essential oils. O. catharinensis became an endangered species mostly because of its intense exploitation and its low seed production and viability. The objective of this work was to study O. catharinensis somatic embryogenesis through the comparative proteome analysis of different embryogenic stages as well as cell aggregates maintained in different culture media that produce distinct cell embryogenic competence. For that, the protein extracts of cell aggregates (calus) cultured in different medias (WPM, MS, MS+2,4-D) and of the somatic embryos in globular (E1), initial cotiledonary (E2), intermediary cotiledonary (E3) and mature embryo (E4) stages were subjected to two-dimensional gel electrophoresis (2-DE). The samples were ground in liquid N2 and glass powder, and the proteins precipitated with TCA/acetone solution followed by extraction and solubilization in 2-DE buffer. The protein concentration was determined using Plus One 2D QuantKit (GE Healthcare). The samples were submitted to isoelectric focusing using pH 4-7 immobilized pH gradient (IPG) strips. We also carried out two-inone gels (Wang et al., 2003) of cell aggregates, where IPG strips with the same pH range for two different samples were loaded side by side on top of a vertical SDS-PAGE gel for separation in the second dimension. After silver staining, the gel images were digitalized and subjected to computational analyses in order to determine the number and intensity of the spots, allowing the determination of protein differential expression. Few protein spots showed different intensities during the development and in the different culture media tested. These spots were analyzed by mass spectrometry using MALDI-TOF and nano-LC MS/MS (LTQ Orbitrap). Just one spot were identified by MALDITOF and eight by LTQ-Orbitrap. Among the identified polypeptides, there were structural proteins like actin, antioxidant proteins like tioredoxin, superoxide dismutase and others like the 14-3-3 family, germin and thiamine biosynthetic enzyme.

ASSUNTO(S)

embriologia - plantas ocotea catharinensis plantas - reprodução embriogênese somática proteômica botanica botânica proteínas - análise

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