RESUMO
Lipoxygenases (LOXs, EC 1.13.11.12) are a class of non-heme iron containing dioxygenases which catalyze the regiospecific and stereospecific hydroperoxidation of polyunsaturated fatty acids with 1,4-pentadiene system such as linoleic acid and linolenic acid in plants. In this work we studied the LOX activity in damaged as well as in distal leaves in response to specialist (Agraulis vanillae vanillae) or generalist (Spodoptera frugiperda) insect attack. Enzymatic assays showed that induction of LOX activity occurred locally and systemically in response to both insects' attacks. Northern blot analysis revealed that LOX expression is also insect-inducible in agreement with enzymatic assay results. In addition, northern analysis corroborated previous reports that LOX activity is wound- and methyl jasmonate-inducible. These results suggest that the herbivore-response in passion fruit is mediated by jasmonates, since a key enzyme of the biosynthetic pathway of jasmonic acid is induced upon lepidopteran insects' attacks.
Assuntos
Lepidópteros/fisiologia , Lipoxigenase/biossíntese , Passiflora/enzimologia , Animais , Ciclopentanos/metabolismo , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica de Plantas , Lipoxigenase/genética , Oxilipinas/metabolismo , Passiflora/genética , Folhas de Planta/enzimologia , RNA de Plantas/metabolismoRESUMO
The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety "Vitória de Verão" genetically modified.
Assuntos
Antígenos de Superfície da Hepatite B/genética , Vacinas contra Hepatite B/genética , Lactuca/genética , Plantas Geneticamente Modificadas/genética , DNA Recombinante , Lactuca/imunologia , Reguladores de Crescimento de Plantas , Plantas Geneticamente Modificadas/imunologia , Plântula/genética , Plântula/imunologia , Vacinas de Plantas ComestíveisRESUMO
The induction of a chloroplast-localized 13-lipoxygenase (13-LOX) in passion fruit leaves in response to methyl jasmonate (MeJa) was previously reported. Since allene oxide synthase (AOS) is a key cytochrome P450 enzyme in the oxylipin pathway leading to AOS-derived jasmonates, the results above led in turn to an investigation of AOS in our model plant. Spectrophotometric assays showed that 24 h exposure of MeJa caused a high increase in 13-hydroperoxy linolenic acid (13-HPOT) metabolizing activity in leaf tissue. Western analysis using polyclonal antibodies against tomato AOS strongly indicate that, at least a part of the 13-HPOT metabolizing capacity can be attributed to AOS activity. We cloned the cDNA from a novel AOS encoding gene from passion fruit, named PfAOS. The 1,512 bp open reading frame of the AOS-cDNA codes a putative protein of 504 amino acid residues containing a chloroplast target sequence. Database comparisons of the deduced amino acid sequence showed highest similarity with dicot AOSs. Immunocytochemistry analysis showed the compartmentalization of AOS in chloroplasts of MeJa treated leaves, corroborating the predicted subcellular localization. Northern analysis showed that AOS gene expression is induced in leaf tissue in response to mechanical wounding and exposure to MeJa. In addition, such treatments caused an increase in papain inhibitor(s) in leaf tissue. Taken together, these results indicate that PfAOS may play an important role in systemic wound response against chewing insect attack. Furthermore, it can be useful as a tool for understanding the regulation of jasmonates biosynthesis in passion fruit.
Assuntos
Acetatos/farmacologia , Cloroplastos/metabolismo , Ciclopentanos/farmacologia , Oxirredutases Intramoleculares/metabolismo , Oxilipinas/farmacologia , Passiflora/metabolismo , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , Cistatinas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Imuno-Histoquímica , Oxirredutases Intramoleculares/genética , Dados de Sequência Molecular , Passiflora/efeitos dos fármacos , Passiflora/genética , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico/efeitos dos fármacos , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety 'Vitória de Verão' genetically modified.