RESUMO
As a first step toward the exploitation of the disaccharide trehalose as a stress-protective and preservative agent in plants, we engineered trehalose biosynthesis in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. In leaves of transgenic tobacco plants, very low levels of trehalose accumulation were obtained (0.11 mg g-1 fresh weight), whereas in transgenic potato tubers, no trehalose accumulated at all. Plant trehalase activity was shown to affect the accumulation of trehalose in these plants. An increase in trehalose accumulation, up to 0.41 and 4.04 mg g-1 fresh weight in tobacco leaves and potato micro-tubers, respectively, was noted when the potent trehalase inhibitor validamycin A was added to in vitro plants and to hydroponically grown greenhouse plants. Stunted growth and the formation of lancet-shaped leaves by trehalose-accumulating tobacco plants suggest a negative effect of trehalose biosynthesis on N. tabacum development. It is surprising that experiments with wild-type plants cultured in the presence of validamycin A indicate that, despite current belief, the capacity to synthesize trehalose may not be restricted to primitive phyla of vascular plants and certain "resurrection plants," but may exist throughout the angiosperms.
Assuntos
Plantas Geneticamente Modificadas/metabolismo , Trealase/antagonistas & inibidores , Trealose/metabolismo , Clonagem Molecular , Escherichia coli/genética , Glucosiltransferases/genética , Inositol/análogos & derivados , Inositol/farmacologia , Monoéster Fosfórico Hidrolases/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Tóxicas , Solanum tuberosum/genética , Nicotiana/genéticaRESUMO
Phytase from Aspergillus niger increases the availability of phosphorus from feed for monogastric animals by releasing phosphate from the substrate phytic acid. A phytase cDNA was constitutively expressed in transgenic tobacco (Nicotiana tabacum) plants. Secretion of the protein to the extracellular fluid was established by use of the signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be approximately 90-fold higher than in total leaf extract, showing that the enzyme was secreted. This was confirmed by use of immunolocalization. Despite differences in glycosylation, specific activities of tobacco and Aspergillus phytase were identical. Phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. Comparison of phytase accumulation and relative mRNA levels showed that phytase stably accumulated in transgenic leaves during plant growth.
Assuntos
6-Fitase/biossíntese , Aspergillus niger/enzimologia , 6-Fitase/análise , 6-Fitase/genética , Sequência de Aminoácidos , Aspergillus niger/genética , Western Blotting , Espaço Extracelular/enzimologia , Expressão Gênica , Glicosídeo Hidrolases , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Folhas de Planta , Plantas Geneticamente Modificadas , Plantas Tóxicas , Plasmídeos , Sinais Direcionadores de Proteínas/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , NicotianaRESUMO
We have used a modified CaMV 35S promoter to direct the expression of chimaeric genes encoding human serum albumin (HSA) in transgenic potato and tobacco plants. To secrete the protein, either the human prepro-sequence or the signal sequence from the extracellular tobacco protein PR-S was used. We demonstrate secretion of HSA with both types of signal sequences in transgenic leaf tissue and in suspension cultures. HSA produced in transgenic potato plants was purified to chromatographic homogeneity. N-terminal amino acid sequence analysis revealed that the processing of the precursor protein was dependent on the type of signal sequence. Expression of the human preproHSA gene lead to partial processing of the precursor and secretion of proHSA. Fusion of HSA to the plant PR-S presequence resulted in cleavage of the presequence at its natural site and secretion of correctly processed HSA that is indistinguishable from the authentic human protein.
Assuntos
Plantas/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Albumina Sérica/biossíntese , Transfecção , Sequência de Aminoácidos , Sequência de Bases , Quimera/genética , Humanos , Dados de Sequência Molecular , Plantas/metabolismo , Plantas Tóxicas , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
1. Cytosol from pig skeletal muscle, but not heart, contains an inhibitor of AMP-deaminase (AMP-D, EC 3.5.4.6) which reduces AMP-D activity 8-fold. 2. Heart and skeletal muscle AMP-D have been purified to apparent homogeneity by cellulose phosphate and DEAE-Sephacel chromatography. 3. AMP-D from skeletal muscle is inhibited more severely than the heart enzyme by an increase in adenylate energy charge to levels exceeding 0.4. Nevertheless both enzymes seem to be regulated by the energy charge, which contrasts with reports for rabbit heart AMP-D.
