Prospection of tissue specific promoters in coffee : [PB620]

The majority of transgenic organisms reported in the literature have been made using constitutive promoters. However, there are economic, environmental and biosecurity related restrictions involving indiscriminate (constitutive) expression of heterologous genes. The usage of tissue-specific and induced promoters can resolve those issues by limiting the expression of a transgene to the necessary tissues and conditions. The promoters currently used at Embrapa are transnational properties, burdening the research and causing technological dependence. Therefore, the objective of this work was to find and characterize tissue and organ specific promoter in Coffea spp. We have used the Coffee genome database in silico tools to find genes preferentially expressed in root, leaf and fruit. In this way we found 72 organ-specific candidates: 18 apparently preferentially expressed on leaves, 14 on roots and 40 on fruits. Some of those candidates where tested in vitro using RT-PCR, semi-quantitative PCR, northern blotting and qPCR assays. All four leaf-specific candidates tested (GCFo1, GCFo2, GCFo3 and GCFo4) and at least one of the two fruit-specific candidates tested (GCFr1 e GCFr2) where confirmed to be preferentially expressed on their respective organs. Temporal and spatial expression assays showed that GCFr2 has its expression peak at the endosperm, 180 days after flowering. The highest expressed genes of leaf (GCFo3 and GCFo4) and fruit (GCFr2) were used as probes to isolate its respective promoter through a BAC libraries screening or using the Genome Walker Universal Kit (Clontech). Results concerning gene expression and the molecular characterization of these genes will be presented. (Resume d'auteur)