Indução do promotor GmNAC6 por elicitores de estresses e mapeamento de regiões reguladoras

• Autor(a) principal: Rosado, Gustavo Leão
• Data de Publicação: 2012
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2445
• topic: GmNAC6; Estresse; Regiões reguladoras; GmNAC6; Stress; Regulatory regions; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

NAC domain-containing proteins are unique to plants and comprise a large gene family of transcriptional factors implicated in diverse aspects of plant development and stress responses. The GmNAC6 was recently identified as a downstream component of the ER (endoplasmic reticulum) stress- and osmotic stress-induced NRP (N-rich proteins)-mediated cell death signaling pathway. This investigation aimed at characterizing the GmNAC6 promoter activity in response to different biotic and abiotic stimuli, focusing on the identification of possible cis-regulatory elements that control GmNAC6 expression. For this, 1-kb 5 flanking sequences of GmNAC6 gene were isolated from the soybean genome, fused to the β-glucuronidase (GUS) reporter gene and introduced in N. tabacum by Agrobacterium-mediated transformation in order to study tissue-specific and stress-induced transcriptional activation of GmNAC6 gene. Primary transformants were monitored by PCR and exposed to different hormones and stress treatments, such as tunicamycin, polyethylene glycol, cycloheximide, BAP, salicylic acid, jasmonic acid and abscisic acid. Gene reporter activity was monitored qualitatively by histochemical analyses and quantitatively by fluorometry. Except for PEG that failed to induce promoter activity in the vascular tissues of stems, all the other treatments promoted a systemic induction of promoter activity in all tissues analyzed, displaying a ubiquitous pattern of cell staining. Nevertheless, the level of expression varied with tissue type. Consistent with the involvement of GmNAC6 in cell death, GUS staining was more prominent in older tissues, except for treatment with ABA that displayed higher levels of promoter activity in young leaves. Likewise, the relative level of GUS expression appeared significantly higher in tissues under differentiation. Deletions of the promoter and in silico analysis of putative cis-elements responsive to stimuli from plant promoters allow the identification of discrete modules on NAC6 promoter harboring putative positive and negative cis-elements potentially capable of interfering with promoter induction. All the tested stimuli induced promoter activity, displaying the highest level of induction to hormones that control senescence and biotic response to pathogens and wounding. Furthermore, the promoter was also induced by NRPs, substantiating the argument that GmNAC6 participates in the ER stress and osmotic stress integrative pathway those results in cell death. GmNAC32, another NAC transfactor identified through interaction with GmNAC6, also induced NAC6 promoter. Taken together, these results indicate that the NAC6 gene is transcriptionally induced by different stresses and hormones through discrete regulatory modules on the promoter that harbor positive and negative cis-elements. Furthermore, the present data confirm that NAC6 acts downstream of NRPs in the cell death stress-induced signaling pathway.