Bioquímica e Biologia Molecular
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11837
Navegar
13 resultados
Resultados da Pesquisa
Item Função bioquímica da via daslipoxigenases em plantas de soja submetidas ao ataque de mosca-branca (Bemisia argentifolii)(Ciência e Agrotecnologia, 2004-03) Silva, Francine Barbosa; Oliveira, Maria Goreti de A.; Brumano, Maria Helena N.; Pires, Christiano Vieira; Almeida, Fabrício Tadeu de; Oliveira, Joel Antônio; Pilon, Anderson Martins; Silva, Carlos Henrique Osório; Moreira, Maurilio AlvesNeste trabalho, avaliou-se a capacidade da planta de soja de uma cultivar comercial, IAC-100, e de um genótipo IAC- 100 TN que apresenta ausência de lipoxigenases nas sementes de responderem ao ataque de mosca-branca (Bemisia argentifolii) pela via das Lipoxigenases. Foi realizada a caracterização cinética do pool de lipoxigenases. Os valores de KMapp decresceram nas plantas atacadas, sugerindo uma alteração no pool de lipoxigenases foliares. Valores similares de KMapp, entre os genótipos, indicam que a remoção de lipoxigenases de sementes não interferiram na expressão de lipoxigenases em folhas. Ocorreu aumento significativo na produção de inibidores de proteases. Com esses resultados, infere-se que a produção de inibidores de proteases está envolvida no mecanismo de defesa de soja ao ataque da mosca-branca.Item Efeito da aplicação foliar de ácidos graxos na "via das lipoxigenases" de plantas de soja(Química Nova, 2002-11) Batista, Rosa Bárbara; Oliveira, Maria Goreti de Almeida; Pires, Christiano Vieira; Lanna, Anna Cristina; Gomes, Maria Regina Araújo; José, Inês Chamel; Piovesan, Newton Deniz; Rezende, Sebastião Tavares de; Moreira, Maurilio AlvesThe involvement of lipoxygenase isozymes in several physiological processes of plants has been described but their role is not well understood and more biochemical studies are needed to elucidate the role of the "Lipoxygenase Pathway" in plant physiology. Thus, the biochemical and kinetic characterization of a lipoxygenases "pool" from soybean leaves was carried out. Two genotypes were used: IAC-100 (a normal variety having lipoxygenases in the seeds) and IAC-100 TN (genetically modified genotype, which is devoid of lipoxygenases in the seeds). The plants were submitted to the application of fatty acids (lipoxygenase substrates) on leaves. The results of the biochemical and kinetic studies of lipoxygenase isozymes from leaves of the two genotypes analysed showed that genetic removal of lipoxygenase from seeds did not affect the response of the plant to the treatment, since both genotypes showed similar results.Item Revisiting the soybean GmNAC superfamily(Frontiers in Plant Science, 2018-12) Melo, Bruno P.; Fraga, Otto T.; Silva, José Cleydson F.; Ferreira, Dalton O.; Brustolini, Otávio J. B.; Carpinetti, Paola A.; Machado, Joao Paulo B.; Reis, Pedro A. B.; Fontes, Elizabeth P. B.The NAC (NAM, ATAF, and CUC) genes encode transcription factors involved with the control of plant morph-physiology and stress responses. The release of the last soybean (Glycine max) genome assembly (Wm82.a2.v1) raised the possibility that new NAC genes would be present in the soybean genome. Here, we interrogated the last version of the soybean genome against a conserved NAC domain structure. Our analysis identified 32 putative novel NAC genes, updating the superfamily to 180 gene members. We also organized the genes in 15 phylogenetic subfamilies, which showed a perfect correlation among sequence conservation, expression profile, and function of orthologous Arabidopsis thaliana genes and NAC soybean genes. To validate our in silico analyses, we monitored the stress-mediated gene expression profiles of eight new NAC-genes by qRT-PCR and monitored the GmNAC senescence-associated genes by RNA-seq. Among ER stress, osmotic stress and salicylic acid treatment, all the novel tested GmNAC genes responded to at least one type of stress, displaying a complex expression profile under different kinetics and extension of the response. Furthermore, we showed that 40% of the GmNACs were differentially regulated by natural leaf senescence, including eight (8) newly identified GmNACs. The developmental and stress-responsive expression profiles of the novel NAC genes fitted perfectly with their phylogenetic subfamily. Finally, we examined two uncharacterized senescence-associated proteins, GmNAC065 and GmNAC085, and a novel, previously unidentified, NAC protein, GmNAC177, and showed that they are nuclear localized, and except for GmNAC065, they display transactivation activity in yeast. Consistent with a role in leaf senescence, transient expression of GmNAC065 and GmNAC085 induces the appearance of hallmarks of leaf senescence, including chlorophyll loss, leaf yellowing, lipid peroxidation and accumulation of H 2 O 2 . GmNAC177 was clustered to an uncharacterized subfamily but in close proximity to the TIP subfamily. Accordingly, it was rapidly induced by ER stress and by salicylic acid under late kinetic response and promoted cell death in planta. Collectively, our data further substantiated the notion that the GmNAC genes display functional and expression profiles consistent with their phylogenetic relatedness and established a complete framework of the soybean NAC superfamily as a foundation for future analyses.Item Complete inventory of soybean NAC transcription factors: Sequence conservation and expression analysis uncover their distinct roles in stress response(Gene, 2009-09-01) Pinheiro, Guilherme L.; Marques, Carolina S.; Costa, Maximiller D.B.L.; Reis, Pedro A. B.; Alves, Murilo S.; Carvalho, Claudine M.; Fietto, Luciano G.; Fontes, Elizabeth P. B.We performed an inventory of soybean NAC transcription factors, in which 101 NAC domain-containing proteins were annotated into 15 different subgroups, showing a clear relationship between structure and function. The six previously described GmNAC proteins (GmNAC1 to GmNAC6) were located in the nucleus and a transactivation assay in yeast confirmed that GmNAC2, GmNAC3, GmNAC4 and GmNAC5 function as transactivators. We also analyzed the expression of the six NAC genes in response to a variety of stress conditions. GmNAC2, GmNAC3 and GmNAC4 were strongly induced by osmotic stress. GmNAC3 and GmNAC4 were also induced by ABA, JA and salinity but differed in their response to cold. Consistent with an involvement in cell death programs, the transient expression of GmNAC1, GmNAC5 and GmNAC6 in tobacco leaves resulted in cell death and enhanced expression of senescence markers. Our results indicate that the described soybean NACs are functionally non-redundant transcription factors involved in response to abiotic stresses and in cell death events in soybean.Item Low linolenic soybeans for biodiesel: Characteristics, performance and advantages(Fuel, 2012-06-16) Santos, Eleonice Moreira; Piovesan, Newton Deniz; Barros, Everaldo Gonçalves de; Moreira, Maurilio AlvesSoybean is one of the main raw materials used for biodiesel production. However, the polyunsaturated fatty acids present in soybean seeds are not desirable for this purpose due to their low oxidative stability. Therefore, it is expected that the use of soybean cultivars with low linolenic acid content for biodiesel production will improve its oxidative stability and the cold filter plugging point (CFPP). This work presents the main characteristics, the advantages and performance of low linolenic acid soybean (LL) as compared to a conventional soybean variety (CO) for biodiesel production. The results showed that LL oil and protein contents were similar to those of CO. Phosphatide concentration was higher in LL oil, while total tocopherol content was lower in relation to CO. With respect to LL biodiesel performance, oxidative stability was much higher than that produced from CO, and the CFPP did not change even with the improved fatty acid profile of LL.Item Development of a method to quantify sucrose in soybean grains(Food Chemistry, 2012-02-15) Teixeira, Arlindo I.; Ribeiro, Lucas F.; Rezende, Sebastião T.; Barros, Everaldo G.; Moreira, Maurílio A.The sucrose content of soybean seeds affects the final flavor of soy-derived products. The aim of this work was to develop a simple, low-cost, spectrophotometric method for sucrose quantification in soybean seeds. To achieve this goal, we combined the action of invertase, an enzyme that hydrolyses sucrose into fructose and glucose, with glucose oxidase, an enzyme widely used for glucose quantification. This system was adapted to ELISA plates, making large-scale analyses possible at low cost, with potential application in routine analyses. To validate this method, sucrose content was determined in seeds of 14 soybean cultivars by this new method, as well as by HPLC and the enzymatic method of Stitt. The correlation coefficients were high and significant between the results obtained with the new method and the HPLC method (r = 0.9766) and the Stiff method (0.9461).Item Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein(Phytochemistry, 2007-01-12) Rocha, Carolina S.; Luz, Dirce F.; Oliveira, Marli L.; Baracat-Pereira, Maria C.; Medrano, Francisco Javier; Fontes, Elizabeth P.B.The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of b-strand structure (42%) over the a-helix (9.9%). The fluorescence emis- sion maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K d ) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K d = 2.79 ± 0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6 M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.Item Processing of soybean products by semipurified plant and microbial α-Galactosidases(Journal of Agricultural and Food Chemistry, 2006-05-12) Falkoski, Daniel L.; Guimarães, Valéria M.; Callegari, Carina M.; Reis, Angélica P.; Barros, Everaldo G. de; Rezende, Sebastião T. deGalactooligosaccharides (GO) are responsible for intestinal disturbances following ingestion of legume-derived products. Enzymatic reduction of GO level in these products is highly desirable to improve their acceptance. For this purpose, plant and microbial semipurified α-galactosidases were used for GO hydrolysis in soybean flour and soy molasses. α-Galactosidases from soybean germinating seeds, Aspergillus terreus, and Penicillium griseoroseum presented maximal activities at pH 4.0−5.0 and 45−65 °C. The KM,app values determined for raffinose by the soybean, A. terreus, and P. griseoroseum α-galactosidases were 3.44, 19.39, and 20.67 mM, respectively. The enzymes were completely inhibited by Ag+ and Hg2+, whereas only soybean enzyme was inhibited by galactose. A. terreus α-galactosidase was more thermostable than the enzymes from the other two sources. This enzyme maintained about 100% of its original activity after 3 h at 60 °C. The microbial α-galactosidases were more efficient for reducing GO in soybean flour and soy molasses than soybean enzyme.Item Quantification of anti-nutritional factors and their correlations with protein and oil in soybeans(Anais da Academia Brasileira de Ciências, 2015-08-31) Bueno, Rafael D.; Borges, Leandro L.; God, Pedro I.V. Good; Piovesan, Newton D.; Teixeira, Arlindo I.; Cruz, Cosme Damião; Barros, Everaldo G. deSoybeans contain about 30% carbohydrate, mainly consisting of non-starch polysaccharides (NSP) and oligosaccharides. NSP are not hydrolyzed in the gastrointestinal tract of monogastric animals. These NSP negatively affect the development of these animals, especially the soluble fraction. This work aimed to establish a method to quantify NSP in soybeans, using high performance liquid chromatography (HPLC), and to estimate correlations between NSP, oligosaccharides, protein and oil. Sucrose, raffinose + stachyose, soluble and insoluble NSP contents were determined by HPLC. Oil and protein contents were determined by near-infrared spectroscopy (NIRS). The soluble PNAs content showed no significant correlation with protein, oil, sucrose and raffinose + stachyose contents, but oligosaccharides showed a negative correlation with protein content. These findings open up the possibility of developing cultivars with low soluble NSP content, aiming to develop feed for monogastric animals.Item Purification and characterization of Aspergillus terreus α-Galactosidases and their use for hydrolysis of Soymilk Oligosaccharides(Applied Biochemistry and Biotechnology, 2011-02-18) Ferreira, Joana Gasperazzo; Reis, Angélica Pataro; Guimarães, Valéria Monteze; Falkoski, Daniel Luciano; Silva Fialho, Lílian da; Rezende, Sebastião Tavares deα-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4–7. Maximal activities were determined at 60, 55, and 50°C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50°C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg2+, Ag+, and Cu2+. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymes