RESUMO
The rolA gene of Agrobacterium rhizogenes contains in its untranslated leader region a spliceosomal intron, which is spliced in Arabidopsis and in Nicotiana tabacum. Expression under the control of the 35S promoter from cauliflower mosaic virus of a rolA gene derivative defective in splicing still causes alterations of growth in transgenic tobacco plants. Splicing of rolA mRNA is required for efficient expression of the rolA phenotype in vivo. Moreover, splicing is required for efficient in vitro translation of the rolA mRNA. In contrast, expression of a 35S-rolA gene derivative with the ATG initiation codon replaced by ATA does not cause any phenotypical alteration. Mutations leading to amino acid substitutions at positions 37 and 40 of the rolA coding region were isolated as null mutants in Arabidopsis plants transgenic for the rolA gene. However, when expressed in tobacco under the control of the 35S promoter, they cause a rolA phenotype reduced in the expressivity of its traits. The molecular characterization of rolA mutants might be useful for understanding the biochemical function of the rolA protein.
Assuntos
Proteínas de Bactérias/genética , Mutação , Nicotiana/genética , Plantas Tóxicas , Precursores de RNA/genética , RNA Mensageiro/genética , Rhizobium/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Análise Mutacional de DNA , Regulação da Expressão Gênica de Plantas , Íntrons , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Mutação Puntual , Splicing de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.
Assuntos
Proteínas Luminescentes/metabolismo , Plantas/genética , Sequência de Bases , Clonagem Molecular , DNA Complementar , Fluorescência , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Splicing de RNARESUMO
The rolA gene encoded on the Ri plasmid A4 of Agrobacterium rhizogenes is one of the transferred (TL-DNA) genes involved in the pathogenesis of hairy-root disease in plants. The function of the 100-amino acid protein product of rolA is unknown, although its expression causes physiological and developmental alterations in transgenic plants. The rolA gene of A. rhizogenes contains an intron in its untranslated leader region that has features typical of plant pre-messenger RNA introns. Transcription and splicing of the rolA pre-messenger RNA occur in the plant cell.
