RESUMO
The protein population of cassava root layers was characterized by SDS-PAGE and bidimensional polyacrylamide gel electrophoresis. SDS-Page revealed the presence of a protein population in the molecular weight range between 94 and 20 kDa. The expression pattern of these proteins was well-defined within the different layers. Partial protein sequence analyses and preliminary results on the layer-specific expression pattern obtained with Northern analyses are presented.
Assuntos
Manihot/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , DNA de Plantas/química , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Amplificação de Genes , Manihot/química , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Raízes de Plantas/química , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Prolaminas , RNA de Plantas/análise , Análise de Sequência de DNARESUMO
The expression of Brazil nut storage albumin genes is highly regulated during seed development. Several sequences in the promoter of one of these genes show homologies with the target sites of the maize O2 bZIP regulatory protein. We therefore asked whether the O2 protein would recognize these promoter sequences. We show that the O2 protein binds to three different sequences (F1, F2 and F3). F1 and F3 are hybrid C/G and A/G boxes, respectively, that are homologous to the O2-binding site of a maize alpha-zein gene. F2 is a new O2-binding sequence related to the O2 target sites of the Coix alpha-coxin, the maize b-32 genes and the AP-1 pseudopalindrome. Molecular modelling showed that an Asn and a Ser in the 02 DNA binding domain make different base-specific contacts with each operator. 5' Promoter deletions of the be2S1 gene showed that the domain containing the O2 target sites F1 and F2 is required for detectable reporter gene expression in transgenic tobacco seeds. Moreover, the homologous coix O2 protein was shown to in situ transactivate the promoter region encompassing the three O2-binding sites F1, F2 and F3. Thus, these sites may be in vivo regulatory sequences mediating activation by bZIP regulatory proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Nozes/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Fatores de Transcrição/metabolismo , Albuminas 2S de Plantas , Sequência de Aminoácidos , Antígenos de Plantas , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Pegada de DNA , Zíper de Leucina , Modelos Moleculares , Dados de Sequência Molecular , Nozes/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Ligação Proteica , Sementes/genética , Sementes/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transformação Genética , Árvores/genética , Árvores/crescimento & desenvolvimentoRESUMO
Brazil nut 2S albumins lack the essential amino acid tryptophan. In order to improve the protein's nutritional value and create a basis for structural investigations, three separate modified Brazil nut 2S albumin genes were constructed. The first mutant contains five consecutive tryptophan codons, while the other two modified genes encode proteins carrying single tryptophan residues at sites that will allow confirmation of the predicted protein structure through fluorescence quenching techniques. The modified genes, under the regulation of the CaMV 35S promoter, were introduced into Nicotiana tabacum. All three modified genes were correctly transcribed and the 2S albumin accumulated in the seeds of transgenic plants.
Assuntos
Albuminas/química , Nozes/química , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/genética , Triptofano/análise , Agrobacterium tumefaciens/genética , Albuminas/genética , Sequência de Bases , Northern Blotting , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica de Plantas/genética , Imuno-Histoquímica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Conformação Proteica , Software , Nicotiana/genética , Transformação Genética/genética , Triptofano/genéticaRESUMO
A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.
Assuntos
Hibridização de Ácido Nucleico , Vírus de Plantas/isolamento & purificação , Sondas RNA , Viroides/isolamento & purificação , Digoxigenina , Estudos de Avaliação como Assunto , Formaldeído , Frutas/virologia , Vírus de Plantas/genética , RNA Viral/análise , RNA Viral/efeitos dos fármacos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Viroides/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
Bean (Phaseolus vulgaris L.) mature embryos were transformed using biolistic methods with a plasmid containing 2S albumin and beta-glucuronidase structural sequences, both under the control of the 35S CaMV promoter. We have shown that chimaeric tissues could be obtained and that both structural sequences were expressed to similar levels.
Assuntos
Fabaceae/genética , Nozes/genética , Proteínas de Plantas/genética , Plantas Medicinais , Albuminas 2S de Plantas , Antígenos de Plantas , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Nozes/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Two genes, BE2S1 and BE2S2, coding for methionine-rich albumins of Brazil nut (Bertholletia excelsa H.B.K.) have been cloned and their sequence determined. The genes are members of a multigene family and one of them, i.e. BE2S1, codes for one of the dominant 2S isoforms. Its expression is highly regulated during seed development and with respect to tissue specificity. Sequence analysis has shown that the genes contain one intron and that the promoter of BE2S1 shows a canonical TATA motif. The transcription initiation site is located 26 nucleotides downstream from the TATA box. Sequence comparison of the promoter regions of 2S genes from Brassica napus, Arabidopsis thaliana and B. excelsa revealed the presence of TGCA palindromic sequence that appear to be arranged in a 2S-specific manner.
Assuntos
Albuminas/genética , Alérgenos , Nozes/genética , Proteínas de Plantas/genética , Albuminas 2S de Plantas , Sequência de Aminoácidos , Antígenos de Plantas , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Íntrons/genética , Dados de Sequência Molecular , Família Multigênica/genética , Regiões Promotoras Genéticas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sementes/crescimento & desenvolvimento , Alinhamento de Sequência , TATA Box/genéticaRESUMO
A trypanosomatid with a choanomastigote stage and, therefore, belonging to the genus Crithidia, was isolated in culture from the alimentary tract of the hemipteran genus Zelus. The trypanosomatid was able to grow at 37 C, a characteristic reported to date from only 2 other members of Crithidia, C hutneri and C. luciliae thermophila. Subsequently, the flagellate was cloned for biochemical studies which involved cleaving of kDNA by restriction endonucleases and analyses of the isoenzyme and histone patterns. In all the attributes revealed by the foregoing methods, the organism from Zelus differed from the latter 2 congeneric species. On these and morphologic grounds, this organism appears to belong in a new species for which the name Crithidia brasiliensis sp. n. is proposed.