RESUMEN
Amylose and amylopectin are the 2 major components of plant storage starch. The rice starch branching enzyme (RBE) plays an important role in the starch components of rice. In the present study, we selected a specific 195-bp segment from the RBE3 gene to construct hairpin DNA, which was driven by an endosperm-specific high molecular weight glutenin promoter to regulate the biosynthesis of starch. An RNA interference plasmid for the RBE3 gene was constructed to form double-stranded RNA. Following Agrobacterium-mediated rice transformation (in the cultivar Zhonghua 11), 41 transgenic plants were identified using PCR and Southern blot analysis. Semi-quantitative real-time PCR revealed that RBE3 gene expression was significantly reduced in immature transgenic seeds. Transgenic rice amylose content had an average increase of 140%. The highest rice amylose content was 47.61% and the growth rate increased 238% compared to the non-transgenic controls. Branching enzyme II activity was notably reduced, and ADP-glucose pyrophosphorylase, soluble starch synthase, isoamylase, and pullulanase enzyme activity was markedly reduced in T3 seeds. Relative enzyme activity change explained the reduction in thousand-grain weight in transgenic plants. The present study indicated that amylose content was negatively correlated with branching enzyme II activity, spike size, and thousand-grain weight.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/genética , Amilosa/metabolismo , Silenciador del Gen , Genes de Plantas , Oryza/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Endospermo/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glicósido Hidrolasas/metabolismo , Isoamilasa/metabolismo , Oryza/enzimología , Oryza/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , ARN Interferente Pequeño/genética , Almidón Sintasa/metabolismoRESUMEN
Rat brain glycogen branching enzyme was partially purified in order to elucidate its mechanism of action. The alpha1,4-alpha1,6-glucan polysaccharide was synthesized using rat brain branching enzyme under two different elongation conditions: Glc-1-P and phosphorylase or UDP-Glc and glycogen synthase. The products obtained demonstrated that the cpolysaccharides synthesized (pattern of the spectra obtained in the presence of Krisman's reagent, lambda max, parameter A and R, % beta-amylolysis and degree of branching) under different incubation times are nearly constant. These results imply that the degree of branching of a polysaccharide depends only on the enzyme specificity.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Encéfalo/enzimología , Absorción , Animales , Glucofosfatos/metabolismo , Fosforilasas/metabolismo , Polisacáridos/biosíntesis , Ratas , Ratas Wistar , beta-Amilasa/metabolismoRESUMEN
Different branching enzymes were partially purified in order to get some insight into their mechanism of action. Each branching enzyme is able to synthesize in vitro at any moment, even during very short incubation times, only one type of alpha 1,4-alpha 1,6 glucopolysaccharide, which possesses unique and specific properties (lambda max; A; R;% of beta-amylolysis;% alpha 1,6). Between two branching points, each branching enzyme will leave, in average, a constant and specific number of glucoses alpha 1,4-linked, which is characteristic of that particular enzyme. Consequently the structure of the polysaccharide will keep the same independently from the moment of synthesis.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/aislamiento & purificación , Animales , Conformación de Carbohidratos , Glucanos/biosíntesis , Glucanos/química , Glucosa/química , Glucosa/metabolismo , Glucógeno/biosíntesis , Glucógeno/química , Técnicas In Vitro , Hígado/enzimología , Modelos Biológicos , Polisacáridos/biosíntesis , Polisacáridos/química , Ratas , Solanum tuberosum/enzimología , Espectrofotometría , Zea mays/enzimologíaRESUMEN
Starch biogenesis in corn endosperm from Flint, Sugary, Waxy, as a function of the grain filling/period was studied. We have differentially identified the initiation from the elongation process. After incubating under unprimed conditions, two glucose radiolabelled protein bands of 39,5 and 36 kDa were obtained. UDP(14C)Glc was the preferred glucosyl donor but also ADP(14C)Glc was. It was additionally found that more than one glucose was transferred to the protein or to the alpha 1,4-glucan linked to protein from UDPGlc. These results were supported by the fact that the glucosylated protein from UDPGlc liberates maltooligosaccharides after alpha- or beta-amylase treatment. The elongation activity in the first steps related to the glucan linked to protein is different from starch synthase. Therefore, we are proposing a model for starch biogenesis where two new transglucosylating enzyme activities are necessary to prepare the primer for starch synthase.
Asunto(s)
Polisacáridos/metabolismo , Almidón/biosíntesis , Zea mays/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Adenosina Trifosfato/farmacología , Glucosiltransferasas/metabolismo , Glicoproteínas/metabolismo , Modelos Biológicos , Almidón Sintasa/metabolismo , Uridina Trifosfato/farmacología , Zea mays/crecimiento & desarrolloRESUMEN
Alteraçöes histopatológicas do fígado em material de biópsia de duas crianças de sete meses a três anos foram descritas e comparadas com as previamente relatadas na literatura. O diagnóstico foi feito com base nas manifestaçöes clínicas, achados de microscopia óptica e, em um dos casos, nos achados ultra-estruturais
Asunto(s)
Humanos , Masculino , Lactante , Preescolar , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo IV/etiología , Hígado/patología , Enfermedad del Almacenamiento de Glucógeno Tipo IV/complicaciones , Enfermedad del Almacenamiento de Glucógeno Tipo IV/diagnósticoRESUMEN
Chemical and biochemical analysis of the polysaccharide, present in rat thymus, indicate that it consists of glucose units alpha-1,4 and alpha-1,6 linked. Electron microscopy reveals the presence of a polysaccharide, similar to the beta-glycogen particles observed in liver and muscle with an average diameter of 20-30 nm. They are located in the cytoplasmic area of T-cells from the cortical region of the thymus. Enzymatic analysis indicates that the beta-particles contain a highly branched glucan with short external chains. Some of the enzymes of glycogen metabolism: synthase, phosphorylase and branching were for the first time partially purified from rat thymus and some of their properties were studied. Therefore, glycogen appeared to be synthesized in rat thymus.
Asunto(s)
Glucógeno/análisis , Timo/análisis , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Animales , Glucógeno/metabolismo , Glucógeno Sintasa/metabolismo , Histocitoquímica , Microscopía Electrónica , Fosforilasas/metabolismo , Ratas , Ratas Endogámicas , Timo/ultraestructuraRESUMEN
1. Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan-degrading enzymes. 2. In 'sweet corn' maize, two separate fractions with (alpha 1,4)glucan: (alpha 1,4)glucan alpha 6-glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above-mentioned conditions. 3. Comparative analyses performed with native (alpha 1,4)-(alpha 1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans. 4. It may be concluded that the branching enzyme is responsible for the specific degree of (alpha 1,6) branch linkages found in the native polysaccharide.