RESUMO
The SaCLCa1 gene, a putative orthologue of AtCLCa, the Arabidopsis thaliana gene encoding a NO3-/H+ antiporter, was cloned from the halophyte Suaeda altissima. It belonged to the CLC family, comprising anionic channels and anion/H+ antiporters. SaCLCa1 ion specificity was studied by heterologous expression of this gene in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encoded the only CLC family protein, the Cl- transporter Gef1p, in undisrupted strains of this organism. For comparison, the function of another recently identified S. altissima CLC family gene, SaCLCc1, was also characterised. Expression of SaCLCc1 in Δgef1 cells restored their ability to grow on selective media. This supported the chloride specificity of this transporter. By contrast, expression of SaCLCa1 did not complement the growth defect phenotype of Δgef1 cells. However, growth of the Δgef1 mutant on the selective media was partially restored when it was transformed with SaCLCa1(C562â¯T), encoding the modified protein SaCLCa1(P188S), in which proline responsible for NO3- selectivity in selective filter was replaced by serine providing chloride selectivity. Quantitative real-time polymerase chain reactions (qRT-PCR) showed that significant induction of SaCLCa1 occurred in the roots of S. altissima when plants were grown on nitrate-deficient medium, while SaCLCc1 activation was observed in S. altissima leaves of plants grown in increasing Cl- concentrations of nutrient solution. These results suggested that SaCLCa1 and SaCLCc1 function as anionic transporters with nitrate and chloride specificities, respectively.
Assuntos
Chenopodiaceae/genética , Canais de Cloreto/genética , Proteínas de Plantas/genética , Plantas Tolerantes a Sal/genética , Sequência de Aminoácidos , Sequência de Bases , Chenopodiaceae/metabolismo , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Microrganismos Geneticamente Modificados/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Plantas Tolerantes a Sal/metabolismo , Alinhamento de SequênciaRESUMO
One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe2+-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn2+. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl- ions.
