RESUMEN
A procedure to obtain endosperm protein extracts was standardized. After confirming the enrichment with nuclear proteins by immunodetection, the protein profiles of extracts from different seed development stages were compared by SDS-PAGE that showed the existence of several differentially expressed proteins.
Asunto(s)
Proteínas Nucleares/aislamiento & purificación , Semillas/metabolismo , Zea mays/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Desnaturalización Proteica , Semillas/química , Semillas/crecimiento & desarrollo , Zea mays/química , Zea mays/crecimiento & desarrolloRESUMEN
Lipid transfer proteins (LTPs) are antimicrobial peptides (AMPs) involved in the defense of plants against pathogens. Our group has previously characterized and purified a LTP from cowpea (Vigna unguiculata (L.) Walp.) seeds which caused the inhibition of growth of fungal pathogens in vitro. The aim of this work was to obtain the cDNA encoding the cowpea LTP and after cloning, to use the cDNA as a probe for studying its expression profile during the development of cowpea seeds. In this work, the N-terminal sequence of the mature LTP peptide from cowpea was used to produce a degenerated oligonucleotide. This primer allowed the amplification of the LTP cDNA by RT-PCR from mRNA of cowpea seeds. The sequence analysis of the cloned cDNA, named VULTP, showed 494 bp which encoded a polypeptide of 91 amino acids. The deduced peptide presented high homology of similarity to plant LTPs of Vigna radiata var. radiate (94%), Prunus domestica (82%) and Zea mays (72%). The expression profile of the VULTP gene in cowpea was analyzed by Northern blot and revealed that the transcript is not accumulated in adult tissues. Conversely, VULTP mRNA is early and strongly accumulated during seed development. The results obtained to seedling of cowpea demonstrate that the VULTP gene presents differential expression in response to different stress. Further studies will be conducted to try to gain better understanding about the physiological role of this gene in cowpea.
Asunto(s)
Proteínas Portadoras/biosíntesis , Fabaceae/metabolismo , Fusarium , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas , Semillas/crecimiento & desarrollo , Adaptación Fisiológica/genética , Proteínas Portadoras/genética , Clonación Molecular , Frío , ADN Complementario/genética , Fabaceae/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Semillas/genéticaRESUMEN
Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.