RESUMO
We have produced human alpha1-antitrypsin (A1AT), a major therapeutic protein, in genetically engineered tobacco plastids. Four different expression vectors have been evaluated which encode A1AT under the control of various 5' and 3' plastid expression elements. The use of heterologous promoter and terminator sequences derived from the corn and soybean plastid genomes leads to simpler and predictable recombinant genome patterns, avoiding unwanted recombination products between introduced and resident tobacco sequences. High level expression of unglycosylated A1AT, representing up to 2% of total soluble proteins, has been measured in leaves of transgenic tobacco lines. Some heterogeneity in the recombinant A1AT is detected after 2D protein separation, but the chloroplast-made protease inhibitors are fully active and bind to porcine pancreatic elastase.
Assuntos
Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Nicotiana/genética , Plantas Geneticamente Modificadas , alfa 1-Antitripsina/biossíntese , alfa 1-Antitripsina/genética , Animais , Eletroforese em Gel Bidimensional , Técnicas Genéticas , Glicosilação , Humanos , Espectrometria de Massas , Elastase Pancreática/química , Plastídeos/química , Plastídeos/metabolismo , Estrutura Terciária de Proteína , Suínos , alfa 1-Antitripsina/químicaRESUMO
We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis).
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Endotoxinas/genética , Vetores Genéticos/genética , Glycine max/genética , Plastídeos/genética , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Bioensaio/métodos , Southern Blotting , Western Blotting , DNA de Plantas/análise , DNA de Plantas/genética , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Expressão Gênica , Vetores Genéticos/química , Proteínas Hemolisinas , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Lepidópteros/efeitos dos fármacos , Lepidópteros/crescimento & desenvolvimento , Dados de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/toxicidade , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Glycine max/metabolismo , Glycine max/toxicidade , Transformação Genética/genética , Transgenes/genéticaRESUMO
We report the complete sequence of a calcium channel alpha 1 subunit cDNA cloned from a Drosophila head cDNA library. This cDNA encodes a deduced protein containing 2516 amino acids with a predicted molecular weight of 276,493. The deduced protein shares many features with vertebrate homologs, including four repeat structures, each containing six transmembrane domains, a conserved ion selectivity filter region between transmembrane domains 5 and 6, and an EF hand in the carboxy tail. The Drosophila subunit has unusually long initial amino and terminal carboxy tails. The region corresponding to the last transmembrane domain (IVS6) and the adjacent cytoplasmic domain has been postulated to form a phenylalkylamine-binding site in vertebrate calcium channels. This region is conserved in the Drosophila sequence, while domains thought to be involved in dihydropyridine binding show numerous changes. The Drosophila subunit exhibits 78.3% sequence similarity to the rat brain type D calcium channel alpha 1 subunit, and so has been designated as a Drosophila melanogaster calcium channel alpha 1 type D subunit (Dmca1D). In situ hybridization shows that Dmca1D is highly expressed in the embryonic nervous system. Northern analysis shows that Dmca1D cDNA hybridizes to three size classes of mRNA (9.5, 10.2, and 12.5 kb) in heads, but only two classes (9.5 and 12.5 kb) in bodies and legs. PCR analysis suggests that the Dmca1D message undergoes alternative splicing with more heterogeneity appearing in head and embryonic extracts than in bodies and legs.
Assuntos
Encéfalo/metabolismo , Canais de Cálcio/metabolismo , Clonagem Molecular , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Ratos/metabolismo , Aminas/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio/classificação , Canais de Cálcio/genética , Clonagem Molecular/métodos , DNA Complementar/genética , Di-Hidropiridinas/farmacologia , Drosophila melanogaster/genética , Hibridização In Situ , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
Exopolygalacturonase (exoPG) is a pectin-degrading enzyme abundant in maize pollen. Using immunochemistry and in situ hybridization it is shown that in addition to its presence in pollen, exoPG is also present in sporophytic tissues, such as the tapetum and mesophyll cells. The enzyme is located in the cytoplasm of pollen and of some mesophyll cells. In other mesophyll cells, the tapetum and the pollen tube, exoPG is located in the cell wall. The measurement of enzyme activity shows that exoPG is ubiquitous in the vegetative organs. These results suggest a general function for exoPG in cell wall edification or degradation. ExoPG is encoded by a closely related multigene family. The regulation of the expression of one of the exoPG genes was analyzed in transgenic tobacco. Reporter GUS activity was detected in anthers, seeds and stems but not in leaves or roots of transgenic plants. This strongly suggests that the ubiquitous presence of exoPG in maize is the result of the expression of different exoPG genes.
Assuntos
Regulação da Expressão Gênica , Glicosídeo Hidrolases/isolamento & purificação , Zea mays/enzimologia , Sequência de Bases , Análise Mutacional de DNA , Glucuronidase/biossíntese , Glucuronidase/genética , Glicosídeo Hidrolases/genética , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Família Multigênica , Plantas Geneticamente Modificadas , Plantas Tóxicas , Pólen/enzimologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Sementes/enzimologia , Deleção de Sequência , Distribuição Tecidual , Nicotiana/genética , Zea mays/genéticaRESUMO
A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg et al. 1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker gene.
RESUMO
A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.