Navegando por Autor "Waclawovsky, Alessandro J."
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Item Combinatorial regulation modules on GmSBP2 promoter: a distal cis-regulatory domain confines the SBP2 promoter activity to the vascular tissue in vegetative organs(Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2006-03-10) Waclawovsky, Alessandro J.; Freitas, Rejane L.; Rocha, Carolina S.; Contim, Luis Antônio S.; Fontes, Elizabeth P.B.The Glycine max sucrose binding protein (GmSBP2) promoter directs phloem-specific expression of reporter genes in transgenic tobacco. Here, we identified cis-regulatory domains (CRD) that contribute with positive and negative regulation for the tissue-specific pattern of the GmSPB2 promoter. Negative regulatory elements in the distal CRD-A (−2000 to −700) sequences suppressed expression from the GmSBP2 promoter in tissues other than seed tissues and vascular tissues of vegetative organs. Deletion of this region relieved repression resulting in a constitutive promoter highly active in all tissues analyzed. Further deletions from the strong constitutive −700GmSBP2 promoter delimited several intercalating enhancer-like and repressing domains that function in a context-dependent manner. Histochemical examination revealed that the CRD-C (−445 to −367) harbors both negative and positive elements. This region abolished promoter expression in roots and in all tissues of stems except for the inner phloem. In contrast, it restores root meristem expression when fused to the −132pSBP2-GUS construct, which contains root meristem expression-repressing determinants mapped to the 44-bp CRD-G (−136 to −92). Thus, the GmSBP2 promoter is functionally organized into a proximal region with the combinatorial modular configuration of plant promoters and a distal domain, which restricts gene expression to the vascular tissues in vegetative organs.Item The soybean sucrose binding protein gene family: genomic organization, gene copy number and tissue‐specific expression of the SBP2 promoter(Journal of Experimental Botany, 2003-12-01) Contim, Luis Antônio S.; Waclawovsky, Alessandro J.; Delú‐Filho, Nelson; Pirovani, Carlos P.; Clarindo, Wellington R.; Loureiro, Marcelo E.; Carvalho, Carlos R.; Fontes, Elizabeth P. B.The sucrose binding protein (SBP) from soybean has been implicated as an important component of the sucrose uptake system. Two SBP genomic clones, gsS641.1 and gsS641.2, which correspond to allelic forms of the GmSBP2/S64 gene, have been isolated and characterized. As a member of the seed storage protein superfamily, it has been shown that the SBP gene structure is similar to vicilin genes with intron/exon boundaries at conserved positions. Fluores cence in situ hybridization (FISH) suggested that the soybean SBP gene family is represented by at least two non‐allelic genes corresponding to the previously isolated GmSBP1 and GmSBP2/S64 cDNAs. These two cDNAs share extensive sequence similarity but are located at different loci in the soybean genome. To investigate transcriptional activation of the GmSBP2 gene, 2 kb 5′‐flanking sequences of gsS641.1 and gsS641.2 were fused to the β‐glucuronidase (GUS) reporter gene and to the green fluorescent protein (GFP) reporter gene and inde pendently introduced into Nicotiana tabacum by Agrobacterium tumefaciens‐mediated transformation. The SBP2 promoter directed expression of both GUS and GFP reporter genes with high specificity to the phloem of leaves, stems and roots. Thus, the overall pattern of SBP–GUS or SBP–GFP expression is consistent with the involvement of SBP in sucrose translocation‐dependent physiological processes.