RESUMO
PLA is one of the most promising bio-compostable and bio-degradable thermoplastic polymers made from renewable sources. PLA is generally produced by ring opening polymerization (ROP) of lactide using the metallic/bimetallic catalyst (Sn, Zn, and Al) or other organic catalysts in a suitable solvent. In this work, reactive extrusion experiments using stannous octoate Sn(Oct)2 and tri-phenyl phosphine (PPh)3 were considered to perform ROP of lactide. Ultrasound energy source was used for activating and/or boosting the polymerization as an alternative energy (AE) source. Ludovic® software, designed for simulation of the extrusion process, had to be modified in order to simulate the reactive extrusion of lactide and for the application of an AE source in an extruder. A mathematical model for the ROP of lactide reaction was developed to estimate the kinetics of the polymerization process. The isothermal curves generated through this model were then used by Ludovic software to simulate the "reactive" extrusion process of ROP of lactide. Results from the experiments and simulations were compared to validate the simulation methodology. It was observed that the application of an AE source boosts the polymerization of lactide monomers. However, it was also observed that the predicted residence time was shorter than the experimental one. There is potentially a case for reducing the residence time distribution (RTD) in Ludovic® due to the 'liquid' monomer flow in the extruder. Although this change in parameters resulted in validation of the simulation, it was concluded that further research is needed to validate this assumption.
RESUMO
Wheat dwarf geminivirus (WDV) is a single-stranded DNA Mastrevirus. The large intergenic region (LIR) of WDV contains cis-acting elements essential for the replication of the genome as well as for the bidirectional transcription of virus genes. The LIR was fused to the GUS (uidA) reporter gene and the WDV viral sense (V-sense) promoter activity derived from the stable integration of that promoter was analysed in transgenic dicot plants. Various dicot species were tested, including Nicotiana tabacum, Nicotiana benthamiana, Arabidopsis thaliana and Cucumis melo. The GUS activity driven by the WDV promoter was also compared to that obtained in plants transformed with the GUS gene controlled by the CaMV 35S promoter as well as two phloem-specific promoters derived from the Arabidopsis thaliana AtSUC2 and AtAHA3 genes. Histochemistry showed that the WDV V-sense promoter consistently induced an expression pattern restricted to the vascular tissues, predominantly in the phloem of all organs. This promoter exhibited levels of GUS activity comparable to that driven by AtSUC2 and AtAHA3 promoters. A vascular expression pattern was observed in the four dicots tested. This was stable during plant development and was not altered following viral infection by an unrelated geminivirus. The uses of such a promoter are discussed regarding the targeting to the phloem of molecules active against vascular pests or pathogens.
RESUMO
cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513-515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.
Assuntos
Endopeptidases/metabolismo , Luteovirus/enzimologia , Mutação/genética , Solanum tuberosum/virologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência de Bases , Capsídeo/biossíntese , Capsídeo/genética , Caulimovirus/genética , Endopeptidases/química , Endopeptidases/genética , Teste de Complementação Genética , Genoma Viral , Luteovirus/genética , Luteovirus/fisiologia , Fases de Leitura Aberta/genética , Folhas de Planta/microbiologia , Folhas de Planta/virologia , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , RNA Viral/biossíntese , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Rhizobium/genética , Serina/genética , Serina/metabolismo , Solanum tuberosum/microbiologia , Replicação ViralRESUMO
Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.