Eugen Silvano Gander

Expression of the 2S albumin from Bertholletia excelsa in Brassica napus

SummaryThe methionine rich 2S albumin seed storage protein of Bertholletia excelsa has been expressed in seeds of Brassica napus (rapeseed). A chimeric gene driven by the soybean lectin 5′ flanking regions was used to produce a fusion protein consisting of the soybean lectin signal peptide and the propeptide of the Brazil nut 2S albumin. Several transgenic plants were studied at the RNA and protein levels; in each case the chimeric gene was expressed and the protein detected at levels ranging from 0.02% to 0.06% of total protein. Transcriptional studies in a particular transgenic plant show that expression of the gene is tissue specific and developmentally regulated during seed maturation. The endogenous napin genes and the introduced gene are regulated differently, with expression of the chimeric gene paralleling that seen when the soybean lectin gene is expressed in other plant species. Western analysis using antibodies to Brazil nut 2S albumins resulted in the detection of a protein whose size is consistent with correct processing of the precursor.

Particle bombardment-mediated transient expression of a Brazil nut methionine-rich albumin in bean (Phaseolus vulgaris L.)

Bean (Phaseolus vulgaris L.) mature embryos were transformed using biolistic methods with a plasmid containing 2S albumin and β-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.

Evidence for a precursor molecule of Brazil nut 2 S seed proteins from biosynthesis and cDNA analysis

SummarySeed storage proteins were extracted from Brazil nut (Bertholletia excelsa H.B.K.) seed embryos at various maturation stages. Salt-soluble and water-soluble proteins (globulins and albumins) were separated by gel chromatography and exhaustive dialysis against water. Both fractions were analysed by one- and two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis revealed that both fractions are unusually high in methionine. The albumins consist of a family of low molecular weight polypeptides that are heterogeneous with respect to pI and are identical to the high methionine 2 S proteins described by Youle and Huang (1981). The biosynthesis of this class of proteins in maturing embryos was followed by in vivo labelling combined with immunological studies. Western blotting with monospecific antibodies against purified 2 S albumins and sequencing of a nearly complete cDNA clone revealed that they are synthesized via a precursor polypeptide.

ACGT and vicilin core sequences in a promoter domain required for seed-specific expression of a 2S storage protein gene are recognized by the opaque-2 regulatory protein

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 α-zein gene. F2 is a new O2-binding sequence related to the O2 target sites of the Coix α-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.

Modified 2S albumins with improved tryptophan content are correctly expressed in transgenic tobacco plants

Brazil nut 2S albumins lack the essential amino acid tryptophan. In order to improve the proteins 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.

Identification of cassava root protein genes

The protein population of cassava root layers was characterized bySDS-PAGE and bidimensional polyacrylamide gel electrophoresis. SDS-Pagerevealed the presence of a protein population in the molecular weight rangebetween 94 and 20 kDa. The expression pattern of these proteins was welldefined within the different layers. Partial protein sequence analyses andpreliminary results on the layer-specific expression pattern obtained withNorthern analyses are presented.

Transgenic plants of ramie (Boehmeria nivea Gaud.) obtained by Agrobacterium mediated transformation

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.

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes

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.

Studies toward the identification of transcription factors in cassava storage root

Fatores de transcricao desempenham importantes funcoes em varios processos fisiologicos. Nos ultimos anos, muitos fatores de transcricao tem sido isolados de plantas, emergindo como poderosas ferramentas na manipulacao de caracteristicas agronomicas. No presente trabalho, iniciamos estudos para isolar fatores de transcricao de mandioca (Manihot esculenta Crantz), importante cultura tropical e subtropical. Nossos resultados revelaram tres tipos de proteinas diferencialmente expressas na raiz de reserva de mandioca (Manihot esculenta Crantz):e imunologicamente relacionadas com o fator de transcricao opaco-2 de milho. Experimentos de Southwestern mostraram duas proteinas capazes de interagir in vitro com uma sequencia de DNA do gene be2S1 de castanha-do-brasil.

Characterization of pearl millet prolamins.

We report the physical-chemical characterization of the major alcohol-soluble proteins present in seeds of pearl millet (Pennisetum glaucum) by SDS-PAGE, bidimensional gel electrophoresis, MALDI-TOF/MS and RP-HPLC. We demonstrate the presence of three major prolamins, called A-, B- and C-pennisetin with mass values around 27, 22 and 12 kDa, respectively. We present partial amino acid sequences of these major proteins, which should allow the posterior isolation of the respective genes.