Composição química e nutrientes em sementes das espécies florestais Pente de Macaco, Flor de Paca, Itaúba, Jatobá e Murici Manso

A considerable number of tree species is the flora in some cases, their fruits and seeds turn out to be good sources of nutrientes. The study aimed to verify the chemical composition of nutrientes in seeds of forest species Pente de Macaco, Flor de Paca, Ita ba, Jatoba and Murici Manso. of forest species of Monkey Comb, Flor de Paca, Ita ba, Jatoba and Murici Manso. The experimental design was randomized with five treatments. Analyses were carried out: moisture content, starch content (mg. g (1)), carbohydrates (mg. g (1)), and proteins (albumin, globulin, prolamin, glutelin) (mg. g (1)) and nutrients (N, P, K, Ca, Mg and S). The results showed that the seeds of Flor de Paca had the highest content of starch and protein of Jatoba is a significant source of carbohydrates and the highest levels of phosphorus, calcium and sulfur. For contents of nitrogen Ita ba showed the highest levels and also obtained satisfactory results for calcium and species Murici Manso had the highest levels of potassium and magnesium.

Influência do molibdênio e do cálcio aplicados via semente nas frações protéicas de amendoim cv.iac 886

Chemical composition of seed displays, in general, the same compounds found in other parts of the plant, and the environment where they grow plants, fertilizer and many other factors are able to change this constitution, increasing or decreasing the amount of certain components. The study aimed to determine the effect of application by seed doses of calcium and molybdenum on protein content of peanut seeds cv.IAC 886. The experimental design was randomized blocks in a factorial design with four replicates per treatment, which are constituted by the combination of molybdenum doses (0, 50, 100 and 150 g ha(-1)) and calcium by seeds (0, 1000, 2000 and 3000 mg L-1). The peanut harvest was done manually. Seeds were removed from the pod manually and individually for each treatment and were taken to the Laboratorio de Genetica de Populacoes e Silvicultura, do Departamento de Fitotecnia, Tecnologia de Alimentos e Socio-Economia, da Faculdade de Engenharia de Ilha Solteira da Universidade Estadual Paulista, where we determined the protein (albumin-Alb, prolamin-PRO, glutelin-GLU and globulin-GLO, mg g(-1). Regardless of the doses used the albumin protein fraction showed the highest in the peanut seeds. The addition of molybdenum resulted in increased seed prolamin content in the peanut seeds. The combination of calcium and molybdenum applied to seeds resulted in increased levels of albumin, globulin and glutelin in the peanut seeds.

Biochemical traits useful for the determination of genetic variation in a natural population of Myracrodruon urundeuva

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fracionamento da proteína e estudo termoanalítico das leguminosas: grão de bico (Cicer arietinum), variedade Cíciero e tremoço branco (Lupinus albus L.)

Pós-graduação em Alimentos e Nutrição - FCFAR

Isolamento, fracionamento e caracterização parcial das proteínas de amêndoa de Baru (Dipteryx alata Vog.)

Pós-graduação em Alimentos e Nutrição - FCFAR

Caracterização química e nutricional da folha de moringa (Moringa oleifera Lam.)

Pós-graduação em Alimentos e Nutrição - FCFAR

Variação genética para os caracteres fisiológicos, bioquímicos e nutricionais em sementes de uma população base de Myracrodruon urundeuva procedente de Aquidauana-MS

Pós-graduação em Agronomia - FEIS

Chemical characteristics and fractionation of proteins from Moringa oleifera Lam. leaves

Moringa oleifera Lam, is a leguminous plant, originally from Asia, which is cultivated in Brazil because of its low production cost. Although some people have used this plant as food, there is little information about its chemical and nutritional characteristics. The objective of this study was to characterise the leaves of M. oleifera in terms of their chemical composition, protein fractions obtained by solubility in different systems and also to assess their nutritional quality and presence of bioactive substances. The whole leaf flour contained 28.7% crude protein, 7.1% fat, 10.9% ashes, 44.4% carbohydrate and 3.0 mg 100 g(-1) calcium and 103.1 mg 100 g(-1) iron. The protein profile revealed levels of 3.1% albumin, 0.3% globulins, 2.2% prolamin, 3.5% glutelin and 70.1% insoluble proteins. The hydrolysis of the protein from leaf flour employing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (ME) resulted in 39.5% and 29.5%, respectively. The total protein showed low in vitro digestibility (31.8%). The antinutritional substances tested were tannins (20.7 mg g(-1)), trypsin inhibitor (1.45 TIU mg g(-1)), nitrate (17 mg g(-1)) and oxalic acid (10.5 mg g(-1)), besides the absence of cyanogenic compounds. beta-Carotene and lutein stood out as major carotenoids, with concentrations of 161.0 and 47.0 mu g g(-1) leaf, respectively. Although M. oleifera leaves contain considerable amount of crude protein, this is mostly insoluble and has low in vitro digestibility, even after heat treatment and chemical attack. In vivo studies are needed to better assess the use of this leaf as a protein source in human feed. (C) 2013 Elsevier Ltd. All rights reserved.

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef)

Background: Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. Results: The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. Conclusions: It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

Uso de marcadores moleculares para la racionalización y evaluación de la calidad en las colecciones de Recursos Fitogenéticos de trigo

Esta tesis tiene dos objetivos generales: el primero, analizar el uso de proteínas del endospermo y SSRs para la racionalización de las colecciones de trigo, y el segundo, estudiar la influencia de las proteínas del endospermo, del año de cultivo y del abonado nitrogenado en la calidad en un grupo de variedades locales españolas. Dentro del primer objetivo, se estudió la diversidad genética de la colección de Triticum monococcum L. (escaña menor), y de una muestra de la colección de Triticum turgidum L. (trigo duro) del CRF-INIA, con 2 y 6 loci de gliadinas, y 6 y 24 SSRs, para la escaña menor y el trigo duro, respectivamente. Ambas colecciones presentaron una gran diversidad genética, con una gran diferenciación entre las variedades y pequeña dentro de ellas. Los loci de gliadinas mostraron una gran variabilidad, siendo los loci Gli-2 los más útiles para distinguir variedades. En la escaña menor, las gliadinas presentaron mayor poder de discriminación que los SSRs; aunque en trigo duro los SSRs identificaron más genotipos. El número de alelos encontrado fue alto; 24 y 38 en gliadinas, y 29 y 203 en SSRs, en escaña menor y trigo duro, respectivamente. En trigo duro, se identificaron 17 alelos nuevos de gliadinas lo que demuestra que el germoplasma español es muy singular. En ambas especies, se detectaron asociaciones entre la variación alélica en prolaminas y el origen geográfico y filogenético de las variedades. La utilidad de las proteínas (6 loci de gliadinas, 2 loci de gluteninas y proteína total) y de los SSRs (24 loci) para verificar duplicados, y analizar la variabilidad intraaccesión, se estudió en 23 casos de duplicados potenciales de trigo duro. Los resultados indicaron que tanto los biotipos como las accesiones duplicadas mostraban el mismo genotipo en gliadinas, pocas diferencias o ninguna en las subunidades de gluteninas HMW y proteína total, y diferencias en menos de tres loci de SSRs. El mismo resultado se obtuvo para los biotipos de la colección de T. monococcum. Sin embargo, las discrepancias observadas en algunos casos entre proteínas y SSRs demostraron la utilidad del uso conjunto de ambos tipos de marcadores. Tanto las proteínas como los SSRs mostraron gran concordancia con los caracteres agro-morfológicos, especialmente cuando las diferencias entre los genotipos eran grandes. Sin embargo, los caracteres agro-morfológicos fueron menos discriminantes que los marcadores moleculares. Para el segundo objetivo de la tesis, se analizó la variación alélica en siete loci de prolaminas relacionados con la calidad en trigo duro: Glu-A1 y Glu-B1 de gluteninas HMW, Glu-A3, Glu-B3 y Glu-B2 de gluteninas B-LMW, y Gli-A1 y Gli-B1 de gliadinas. La submuestra analizada incluía variedades locales de todas las provincias españolas donde se ha cultivado tradicionalmente el trigo duro. Todos los loci, excepto el Glu-B2, mostraron gran variabilidad genética, siendo los Glu-3 los más polimórficos. En total, se identificaron 65 alelos, de los que 29 eran nuevos, que representan una fuente importante de variabilidad genética para la mejora de la calidad. Se detectaron diferencias en la composición en prolaminas entre la convar. turgidum y la zona norte, y la convar. durum y la zona sur; el genotipo Glu-B3new-1 - Gli-B1new-1 fue muy común en la convar. turgidum, mientras que el Glu-B3a - Gli-B1c, asociado con mejor calidad, fue más frecuente en la convar. durum. En la convar. turgidum, se observó mayor variabilidad que en la convar. durum, principalmente en los loci Glu-B1 y Glu-B3, lo que indica que esta convariedad puede ser una fuente valiosa de nuevos alelos de gluteninas. Esta submuestra fue evaluada para calidad (contenido en proteína, P, y test de sedimentación, SDSS) con dos dosis de abonado nitrogenado (N), y en dos años diferentes. No se detectaron interacciones Variedad × Año, ni Variedad × N en la calidad. Para la P, los efectos ambientales (año y N) fueron mayores que el efecto de la variedad, siendo, en general, mayor la P con dosis altas de N. La variedad influyó más en el test SDSS, que no se vio afectado por el año ni el N. El aumento del contenido en proteína no influyó significativamente sobre la fuerza del gluten estimada con el SDSS. Respecto a la influencia de las prolaminas en la fuerza del gluten, se confirmó la superioridad del Glu-B3a; aunque también se detectó una influencia alta y positiva de los alelos nuevos Glu-A3new-1, y Glu-B3new-6 y new-9. La no correlación entre el rendimiento (evaluado en un trabajo anterior) y la P, en las variedades adaptadas a bajo N, permitió seleccionar cuatro variedades locales con alto rendimiento y buena fuerza del gluten para producción con bajo N. SUMMARY There are two main objectives in this thesis: The first, to analyse the use of endosperm proteins and SSRs to rationalize the wheat collections, and the second, to study the influence on quality of endosperm proteins, year and nitrogen fertilization in a group of Spanish landraces. For the first objective, we studied the genetic diversity of the collection of Triticum monococcum L. (cultivated einkorn), and of a sample of the collection of Triticum turgidum L. (durum wheat) maintained at the CRF-INIA. Two and 6 gliadin loci, and 6 and 24 SSRs, were used for einkorn and durum wheat, respectively. Both collections possessed a high genetic diversity, being the differentiation large between varieties and small within them. Gliadin loci showed great variability, being the loci Gli-2 the most useful for distinguish among varieties. In einkorn, the gliadins showed higher discrimination power than SSRs; although SSRs identified more genotypes in durum wheat. Large number of alleles were found; 24 and 38 in gliadins, and 29 and 203 in SSRs, for einkorn and durum wheat, respectively. In durum wheat, 17 new alleles of gliadins were identified, which indicate that Spanish durum wheat germplasm is rather unique. Some associations between prolamin alleles and geographical and phylogenetic origin of varieties were found in both species. The value of endosperm proteins (6 gliadin loci, 2 glutenin loci and total protein) and SSRs (24 loci) for validation of duplicates, and monitoring the intra-accession variability, was studied in 23 potential duplicates of durum wheat. The results indicated that biotypes and duplicated accessions showed identical gliadin genotype, few or none differences in HMW glutenin subunits and total protein, and less than three different SSR loci. A similar result was obtained for biotypes of T. monococcum. However, the discrepancies in some cases support the convenience to use together both marker systems. A good concordance among endosperm proteins, agro-morphological traits and SSRs were also found, mainly when differences between genotypes were high. However, agro-morphological traits discriminated less between accessions than molecular markers. For the second objective of the thesis, we analysed the allelic variation at seven prolamin loci, involved in durum wheat quality: Glu-A1 and Glu-B1 of HMW glutenin, Glu-A3, Glu-B3 and Glu-B2 of B-LMW glutenin, and Gli-A1 and Gli-B1 of gliadin. The subsample analysed included landraces from all the Spanish provinces where the crop was traditionally cultivated. All the loci, except for Glu-B2, showed high genetic variability, being Glu-3 the most polymorphic. A total of 65 alleles were studied, 29 of them being new, which represent an important source of variability for quality improvement. Differences in prolamin composition were detected between convar. turgidum and the North zone, and the convar. durum and the South zone; the genotype Glu-B3new-1 - Gli-B1new-1 was very common in the convar. turgidum, while the Glu- B3a - Gli-B1c, associated with better quality, was more frequent in the convar. durum. Higher variability was detected in the convar. turgidum than in the convar. durum, mainly at the Glu-B1 and Glu-B3, showing that this convariety could be a valuable source of new glutenin alleles. The subsample was evaluated for quality (protein content, P, and sedimentation test, SDSS) with two doses of nitrogen fertiliser (N), and in two different years. No significant Variety x Year or Variety x Nitrogen interactions were detected. For P, environmental (year and N) effects were higher than variety effect, being P values , in general, larger with high dose of N. The variety exhibited a strong influence on SDSS test, which was not affected by year and N. Increasing values of P did not significantly influence on gluten strength, estimated with the SDSS. Respect to the prolamin effects on gluten strength, the superiority of Glu-B3a was confirmed; although a high positive effect of the new alleles Glu-A3new-1, and Glu-B3new-6 and new-9 was also detected. The no correlation between yield (evaluated in a previous research) and P, in the landraces adapted to low N, allowed to select four landraces with high yield and high gluten strength for low N production.

Estudio molecular de gluteninas de alto y bajo peso molecular en Triticum aestivum ssp. vulgare L. y su relación con la calidad panadera

El trigo blando (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) presenta propiedades viscoélasticas únicas debidas a la presencia en la harina de las prolaminas: gluteninas y gliadinas. Ambos tipos de proteínas forman parte de la red de gluten. Basándose en la movilidad en SDS-PAGE, las gluteninas se clasifican en dos grupos: gluteninas de alto peso molecular (HMW-GS) y gluteninas de bajo peso molecular (LMW-GS). Los genes que codifican para las HMW-GS se encuentran en tres loci del grupo 1 de cromosomas: Glu-A1, Glu-B1 y Glu-D1. Cada locus codifica para uno o dos polipéptidos o subunidades. La variación alélica de las HMW-GS es el principal determinante de de la calidad harino-panadera y ha sido ampliamente estudiado tanto a nivel de proteína como de ADN. El conocimiento de estas proteínas ha contribuido sustancialmente al progreso de los programas de mejora para la calidad del trigo. Comparadas con las HMW-GS, las LMW-GS forman una familia proteica mucho más compleja. La mayoría de los genes LMW se localizan en el grupo 1 de cromosomas en tres loci: Glu-A3, Glu-B3 y Glu-D3 que se encuentran estrechamente ligados a los loci que codifican para gliadinas. El número de copias de estos genes ha sido estimado entre 10-40 en trigo hexaploide, pero el número exacto aún se desconoce debido a la ausencia de un método eficiente para diferenciar los miembros de esta familia multigénica. La nomenclatura de los alelos LMW-GS por electroforesis convencional es complicada, y diferentes autores asignan distintos alelos a la misma variedad lo que dificulta aún más el estudio de esta compleja familia. El uso de marcadores moleculares para la discriminación de genes LMW, aunque es una tarea dificil, puede ser muy útil para los programas de mejora. El objetivo de este trabajo ha sido profundizar en la relación entre las gluteninas y la calidad panadera y desarrollar marcadores moleculares que permitan ayudar en la correcta clasificación de HMW-GS y LMW-GS. Se han obtenido dos poblaciones de líneas avanzadas F4:6 a partir de los cruzamientos entre las variedades ‘Tigre’ x ‘Gazul’ y ‘Fiel’ x ‘Taber’, seleccionándose para los análisis de calidad las líneas homogéneas para HMW-GS, LMW-GS y gliadinas. La determinación alélica de HMW-GS se llevó a cabo por SDS-PAGE, y se complementó con análisis moleculares, desarrollándose un nuevo marcador de PCR para diferenciar entre las subunidades Bx7 y Bx7*del locus Glu-B1. Resumen 2 La determinación alélica para LMW-GS se llevó a cabo mediante SDS-PAGE siguiendo distintas nomenclaturas y utilizando variedades testigo para cada alelo. El resultado no fue concluyente para el locus Glu-B3, así que se recurrió a marcadores moleculares. El ADN de los parentales y de los testigos se amplificó usando cebadores diseñados en regiones conservadas de los genes LMW y fue posteriormente analizado mediante electroforesis capilar. Los patrones de amplificación obtenidos fueron comparados entre las distintas muestras y permitieron establecer una relación con los alelos de LMW-GS. Con este método se pudo aclarar la determinación alélica de este locus para los cuatro parentales La calidad de la harina fue testada mediante porcentaje de contenido en proteína, prueba de sedimentación (SDSS) y alveógrafo de Chopin (parámetros P, L, P/L y W). Los valores fueron analizados en relación a la composición en gluteninas. Las líneas del cruzamiento ‘Fiel’ x ‘Taber’ mostraron una clara influencia del locus Glu-A3 en la variación de los valores de SDSS. Las líneas que llevaban el nuevo alelo Glu-A3b’ presentaron valores significativamente mayores que los de las líneas con el alelo Glu-A3f. En las líneas procedentes del cruzamiento ‘Tigre ’x ‘Gazul’, los loci Glu-B1 y Glu-B3 loci mostraron ambos influencia en los parámetros de calidad. Los resultados indicaron que: para los valores de SDSS y P, las líneas con las HMW-GS Bx7OE+By8 fueron significativamente mejores que las líneas con Bx17+By18; y las líneas que llevaban el alelo Glu-B3ac presentaban valores de P significativamente superiores que las líneas con el alelo Glu-B3ad y significativamente menores para los valores de L . El análisis de los valores de calidad en relación a los fragmentos LMW amplificados, reveló un efecto significativo entre dos fragmentos (2-616 y 2-636) con los valores de P. La presencia del fragmento 2-636 estaba asociada a valores de P mayores. Estos fragmentos fueron clonados y secuenciados, confirmándose que correspondían a genes del locus Glu-B3. El estudio de la secuencia reveló que la diferencia entre ambos se hallaba en algunos SNPs y en una deleción de 21 nucleótidos que en la proteína correspondería a un InDel de un heptapéptido en la región repetida de la proteína. En este trabajo, la utilización de líneas que difieren en el locus Glu-B3 ha permitido el análisis de la influencia de este locus (el peor caracterizado hasta la fecha) en la calidad panadera. Además, se ha validado el uso de marcadores moleculares en la determinación alélica de las LMW-GS y su relación con la calidad panadera. Summary 3 Bread wheat (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) flour has unique dough viscoelastic properties conferred by prolamins: glutenins and gliadins. Both types of proteins are cross-linked to form gluten polymers. On the basis of their mobility in SDS-PAGE, glutenins can be classified in two groups: high molecular weight glutenins (HMW-GS) and low molecular weight glutenins (LMW-GS). Genes encoding HMW-GS are located on group 1 chromosomes in three loci: Glu-A1, Glu-B1 and Glu-D1, each one encoding two polypeptides, named subunits. Allelic variation of HMW-GS is the most important determinant for bread making quality, and has been exhaustively studied at protein and DNA level. The knowledge of these proteins has substantially contributed to genetic improvement of bread quality in breeding programs. Compared to HMW-GS, LMW-GS are a much more complex family. Most genes encoded LMW-GS are located on group 1 chromosomes. Glu-A3, Glu-B3 and Glu-D3 loci are closely linked to the gliadin loci. The total gene copy number has been estimated to vary from 10–40 in hexaploid wheat. However, the exact copy number of LMW-GS genes is still unknown, mostly due to lack of efficient methods to distinguish members of this multigene family. Nomenclature of LMW-GS alleles is also unclear, and different authors can assign different alleles to the same variety increasing confusion in the study of this complex family. The use of molecular markers for the discrimination of LMW-GS genes might be very useful in breeding programs, but their wide application is not easy. The objective of this work is to gain insight into the relationship between glutenins and bread quality, and the developing of molecular markers that help in the allele classification of HMW-GS and LMW-GS. Two populations of advanced lines F4:6 were obtained from the cross ‘Tigre’ x ‘Gazul’ and ‘Fiel’ x ‘Taber’. Lines homogeneous for HMW-GS, LMW-GS and gliadins pattern were selected for quality analysis. The allele classification of HMW-GS was performed by SDS-PAGE, and then complemented by PCR analysis. A new PCR marker was developed to undoubtedly differentiate between two similar subunits from Glu-B1 locus, Bx7 and Bx7*. The allele classification of LMW-GS was initially performed by SDS-PAGE following different established nomenclatures and using standard varieties. The results were not completely concluding for Glu-B3 locus, so a molecular marker system was applied. DNA from parental lines and standard varieties was amplified using primers designed in conserved domains of LMW genes and analyzed by capillary electrophoresis. The pattern of amplification products obtained was compared among samples and related to the protein allele classification. It was possible to establish a correspondence between specific amplification products and almost all LMW alleles analyzed. With this method, the allele classification of the four parental lines was clarified. Flour quality of F4:6 advanced lines were tested by protein content, sedimentation test (SDSS) and alveograph (P, L, P/L and W). The values were analyzed in relation to the lines prolamin composition. In the ‘Fiel’ x ‘Taber’ population, Glu-A3 locus showed an influence in SDSS values. Lines carrying new allele Glu-A3b’, presented a significantly higher SDSS value than lines with Glu-A3f allele. In the ‘Tigre ’x ‘Gazul’ population, the Glu-B1 and Glu-B3 loci also showed an effect in quality parameters, in SDSS, and P and L values. Results indicated that: for SDSS and P, lines with Bx7OE+By8 were significantly better than lines with Bx17+By18; lines carrying Glu-B3ac allele had a significantly higher P values than Glu-B3ad allele values. lines with and lower L The analysis of quality parameters and amplified LMW fragments revealed a significant influence of two peaks (2-616 y 2-636) in P values. The presence of 2-636 peak gave higher P values than 2-616. These fragments had been cloned and sequenced and identified as Glu-B3 genes. The sequence analysis revealed that the molecular difference between them was some SNPs and a small deletion of 21 nucleotides that in the protein would produce an InDel of a heptapeptide in the repetitive region. In this work, the analysis of two crosses with differences in Glu-3 composition has made possible to study the influence of LMG-GS in quality parameters. Specifically, the influence of Glu-B3, the most interesting and less studied loci has been possible. The results have shown that Glu-B3 allele composition influences the alveograph parameter P (tenacity). The existence of different molecular variants of Glu-B3 alleles have been assessed by using a molecular marker method. This work supports the use of molecular approaches in the study of the very complex LMW-GS family, and validates their application in the analysis of advanced recombinant lines for quality studies.

A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter

To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters were isolated and studied using a transient expression analysis system. An oat globulin promoter (AsGlo1) capable of driving strong ES-specific expression in barley and wheat was identified. Progressive 5' deletions and cis element mutations demonstrated that the mechanism of specificity in the AsGlo1 promoter was distinct from that observed in glutelin and prolamin promoters. A novel interrupted palindromic sequence, ACATGTCAT-CATGT, was required for ES specificity and substantially contributed to expression strength of the AsGlo1 promoter. This sequence was termed the endosperm specificity palindrome (ESP) element. The GCN4 element, which has previously been shown to be required for ES specificity in cereal SSP promoters, had a quantitative role but was not required for tissue specificity. The 960-bp AsGlo1 promoter and a 251-bp deletion containing the ESP element also drove ES-specific expression in stably transformed barley. Reporter gene protein accumulated at very high levels (10% of total soluble protein) in ES tissues of plants transformed with an AsGlo1:GFP construct. Expression strength and tissue specificity were maintained over five transgenic generations. These attributes make the AsGlo1 promoter an ideal promoter for biotechnology applications. In conjunction with previous findings, our data demonstrate that there is more than one genetically distinct mechanism by which ES specificity can be achieved in cereal SSP promoters, and also suggest that there is redundancy between transcriptional and post-transcriptional tissue specificity mechanisms in cereal globulin genes.

Feijões: características químicas e ocorrência de aflatoxinas

As leguminosas, como o feijão, são consideradas importantes fontes de nutrientes para humanos e a contaminação por fungos e consequente produção de micotoxinas pode estar diretamente influenciada pela sua composição química. Alguns compostos estão associados aos mecanismos de defesa das leguminosas atuando como inibidores de enzimas digestivas ou barreiras físicas à patógenos. É o caso dos compostos fenólicos (CF) e algumas estruturas de caráter proteico. O objetivo deste estudo foi verificar a susceptibilidade de feijões à contaminação por aflatoxinas (AFLAs), através da avaliação da presença de compostos inibidores de enzimas fúngicas. Foi realizada a validação de um método para determinação de AFLAs em feijão. Os CF livres (solúveis em metanol), conjugados (solúveis em etanol) e ligados, bem como as diferentes frações proteicas (albumina, globulina, glutelina e prolamina) foram determinadas em 10 amostras de feijão pertencentes às espécies Phaseolus vulgaris, Vigna unguiculata e Vigna angularis. O seu potencial como inibidor de α-amilase foi testado nos extratos fenólicos e protéicos. Os feijões vermelho e carioca apresentaram os maiores teores de CF totais (1766 µg.g -1 e 1190 µg.g -1 , respectivamente) e os feijões fradinho e branco os menores teores (183 µg.g -1 e 192 µg.g -1 ). Os extratos de CF conjugados apresentaram os teores mais elevados de AF, onde os feijões amendoim se destacou pela maior concentração (68 µg.g -1 ) e o feijão azuki pelo menor (28 µg.g -1 ). Nos extratos de CF livres e conjugados, o ácido clorogênico foi o majoritário em 60% dos feijões analisados e nos extratos de CF ligados, o ácido ferúlico foi o majoritário em 90% dos feijões analisados. Com relação às frações proteicas solúveis, o feijão carioca apresentou o maior teor de albumina (559 mg.g -1 ), globulina (164 mg.g -1 ) e glutelina (325 mg.g -1 ). Com relação à fração prolamina, o feijão preto (brasileiro e chinês) apresentou o maior teor (64 e 65 mg.g -1 , respectivamente), seguido pelo feijão carioca (54 mg.g -1 ). Os limites de detecção (LDm) obtidos para o método de determinação de AFLAs foram de 2,4 µg.kg-1 ; 0,036 µg.kg-1 e 0,06 µg.kg-1 para as AFLAs B1, B2 e G2 e os limites de quantificação (LQm) foram de 4,8 µg.kg-1 (AFLAB1); 0,12 µg.kg- 1 (AFLA B2 e G2). Não foram detectadas AFLAs B1, B2, G1 e G2 nos feijões analisados. Os CF dos extratos etanólicos dos feijões amendoim e azuki e os extratos contendo as proteínas solúveis em etanol dos feijões carioca e fradinho foram testados quanto ao seu potencial para inibição da α-amilase de Aspergillus oryzae (atividade de 4,8 mg amido hidrolisado.mg proteína-1 .mL-1 ). O extrato proteico do feijão fradinho se destacou, pois atingiu um percentual de inibição específica de aproximadamente 56%. Os CF apresentaram uma tendência à inibição incompetitiva e os extratos proteicos não apresentaram um comportamento de inibição que permitisse definir o mecanismo de inibição. Os extratos protéicos e fenólicos dos feijões mostraram ser capazes de inibir a amilase fúngica sugerindo que este fato pode estar associado a ausência da presença de AFLAs nas amostras analisadas.