RESUMO
Cecropin A is a natural antimicrobial peptide that exhibits fast and potent activity against a broad spectrum of pathogens and neoplastic cells, and that has important biotechnological applications. However, cecropin A exploitation, as for other antimicrobial peptides, is limited by their production and purification costs. Here, we report the efficient production of this bioactive peptide in rice bran using the rice oleosin 18 as a carrier protein. High cecropin A levels were reached in rice seeds driving the expression of the chimeric gene by the strong embryo-specific oleosin 18 own promoter, and targeting the peptide to the oil body organelle as an oleosin 18-cecropin A fusion protein. The accumulation of cecropin A in oil bodies had no deleterious effects on seed viability and seedling growth, as well as on seed yield. We also show that biologically active cecropin A can be easily purified from the transgenic rice seeds by homogenization and simple flotation centrifugation methods. Our results demonstrate that the oleosin fusion technology is suitable for the production of cecropin A in rice seeds, which can potentially be extended to other antimicrobial peptides to assist their exploitation.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Gotículas Lipídicas/química , Oryza/metabolismo , Sementes/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/genética , Genoma de Planta , Espectrometria de Massas , Dados de Sequência Molecular , Oryza/genética , Fenótipo , Óleos de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Sementes/genética , TransgenesRESUMO
Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.
Assuntos
Magnaporthe/patogenicidade , Mariposas/patogenicidade , Oryza/enzimologia , Doenças das Plantas/imunologia , Inibidores de Proteases/metabolismo , Animais , Bacillus thuringiensis/genética , Resistência à Doença , Vírus da Febre Aftosa/genética , Expressão Gênica , Oryza/genética , Oryza/imunologia , Controle Biológico de Vetores , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão , Solanum tuberosum/genética , Transgenes , Zea mays/genéticaRESUMO
In most plants, sucrose is the primary product of photosynthesis, the transport form of assimilated carbon, and also one of the main factors determining sweetness in fresh fruits. Traditional methods for sugar quantification (mainly sucrose, glucose and fructose) require obtaining crude plant extracts, which sometimes involve substantial sample manipulation, making the process time-consuming and increasing the risk of sample degradation. Here, we describe and validate a fast method to determine sugar content in intact plant tissue by using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR). The HR-MAS NMR method was used for quantifying sucrose, glucose and fructose in mesocarp tissues from melon fruits (Cucumis melo var. reticulatus and Cucumis melo var. cantalupensis). The resulting sugar content varied among individual melons, ranging from 1.4 to 7.3 g of sucrose, 0.4-2.5 g of glucose; and 0.73-2.83 g of fructose (values per 100 g fw). These values were in agreement with those described in the literature for melon fruit tissue, and no significant differences were found when comparing them with those obtained using the traditional, enzymatic procedure, on melon tissue extracts. The HR-MAS NMR method offers a fast (usually <30 min) and sensitive method for sugar quantification in intact plant tissues, it requires a small amount of tissue (typically 50 mg fw) and avoids the interferences and risks associated with obtaining plant extracts. Furthermore, this method might also allow the quantification of additional metabolites detectable in the plant tissue NMR spectrum.
Assuntos
Carboidratos/análise , Cucumis melo/química , Espectroscopia de Ressonância Magnética/métodos , Ensaios Enzimáticos , Frutose/análise , Frutas/química , Glucose/análise , Prótons , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sacarose/análise , Fatores de Tempo , Água/análiseRESUMO
14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses.
Assuntos
Suscetibilidade a Doenças/imunologia , Oryza/imunologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Estresse Fisiológico/fisiologia , Zea mays/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , DNA Complementar/genética , Suscetibilidade a Doenças/microbiologia , Secas , Fusarium/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Magnaporthe/fisiologia , Cebolas/genética , Cebolas/metabolismo , Oryza/genética , Oryza/microbiologia , Oryza/fisiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , RNA de Plantas/genética , Proteínas Recombinantes/isolamento & purificação , Plântula/genética , Plântula/imunologia , Plântula/microbiologia , Plântula/fisiologia , Transdução de Sinais/fisiologia , Cloreto de Sódio/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.