Identificação de regiões no promotor do gene SBP2 (sucrose binding protein) de soja que conferem expressão espacial específica

• Autor(a) principal: Freitas, Rejane do Livramento
• Data de Publicação: 2007
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/4350
• topic: Sucrose binding protein; Promotor; Tecido-especificidade; Sucrose binding protein; Promotor; Tissue-specific; CNPQ::CIENCIAS BIOLOGICAS

The soybean SBP2 (sucrose binding protein) promoter is capable to drive vascular tissue-specific expression of reporter genes in tobacco transgenic lines. This vascular-specific activity of the SBP2 promoter is confined to a distal region (-2000 to -700 sequences) designated CRD-A (cis-regulatory domain-A). Here, we first confirmed the tissue-specific activity of CRD-A through gain-of-function experiments, in which the CRD-A sequences were directly fused to 5´end of GUS cDNA, -136pSBP2-GUS and -92pSBP2-GUS constructs. In tobacco, CRD-A was able to reduce GUS activity in all organs analyzed, recapitulating in some cases the tissue-specific pattern of the full promoter. In addition, CRD-A promoted GUS transcription in the absence of the proximal TATA-containing region, which suggests that CRD-A may contain cis-regulatory elements to sustain basal transcription. In fact, this region (-2000 a -700) harbors several TATA box-like sequences, positions -790, -783 and -761, that potentially may function as alternative TATA boxes. To delimit the cis-regulatory elements responsible for the tissue-specific activity of SBP2 promoter, the -2000 to -700 sequence was divided into five fragments, which were fused to -92pSBP2-GUS construct and used to obtain transgenic lines. Histochemical analysis revealed that all the CRDA sub-fragments reduced the SBP2 promoter activity, as their fusion to the 5 end of -92pSBP2 altered its constitutive expression pattern. These results identified the presence of several potentially cis-regulatory domains. The region encompassing the sequences -1765 to -945 may contain strong shoot apex expression-repressing elements, capable to totally abolish expression, whereas the region -944 to -705 may harbor weaker repressing elements that restricted GUS expression to the vascular tissue. We also found several root expression silencers, operating in the root meristem (-1765 to -705) and in the root elongation zone (-1765 to -1485 and -1211 to -945). Furthermore, the region delimited by positions -1765 to -1485 also exhibited a strong root expressionrepressing element whose effect may be attenuated by cis-regulatory elements present in the -1485 to -705 region. Finally, a cis-element that confines GUS expression to the inner phloem of stem was identified in the region delimited by positions -1485 to -1212. The function of the identified cis-elements was evaluated through electrophoretic mobility shift assay (EMSA) that revealed sequence-specific interactions between putative transfactors from soybean and tobacco nuclear extracts and the -1765/-1485 (fragII) fragment from GmSBP2. To determine whether the SBP2 protein accumulation correlated with the tissuespecific promoter activity, a SBP2-GFP fusion was expressed in tobacco transgenic lines under the control of SBP2 promoter. Fluorescence analysis revealed that the SBP2 protein was, indeed, located in the vascular tissue, which was consistent with SBP2 promoter activity and the involvement of SBP2 in physiological process dependent of sucrose translocation.