Caracterização molecular de genes que codificam a proteína BiP de soja e análise funcional de seus promotores
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2001-08-09
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Universidade Federal de Viçosa
Resumo
A proteína BiP (Binding Protein) está envolvida no dobramento correto de proteínas secretórias e retenção no RE de proteínas dobradas ou montadas incorretamente. BiP também atua auxiliando na solubilização de agregados protéicos durante períodos de estresses. Dois clones de BiP de soja, denominados gsBiP6 e gsBiP9, foram isolados a partir das bibliotecas genômicas provenientes dos fagos λEMBL3 e λZAP II, respectivamente. Os promotores de gsBiP6 e gsBiP9 possuem cis-elementos gerais, característicos de promotores de genes de plantas, como as seqüências de transcrição CAAT e TATA, e cis-elementos que respondem a estresses do retículo endoplasmático (ERSE), além de elementos G-box. Os promotores de BiP foram fusionados ao gene repórter gus e usados para transformar Nicotiana tabacum via A. tumefaciens. Análises histoquímicas de plantas transformadas com as construções -358pbip6-gus ou -2200pbip9-gus revelaram que os promotores de BiP produziram um padrão uniforme de expressão de GUS em folhas, caules e raízes. Intensa atividade histoquímica de GUS foi observada nos feixes vasculares de folhas, caule e raízes e em regiões de traço foliar, assim como, no meristema apical, local onde ocorre intensa divisão celular. Este padrão de distribuição espacial da atividade de GUS sugere estar correlacionado com uma expressão desses genes em tecidos que apresentam alta atividade celular secretória e alta proporção de células em um processo ativo de divisão celular. Coerente com esta observação, a atividade histoquímica de GUS é muito menos intensa no parênquima xilemático. Deleções do promotor de gsBiP6 foram conduzidas na orientação 5’-3’ (- 138pbip6-gus) e no interior do clone Δ(-226/-82)pbip6-gus. Análise dessas deleções em plantas transformadas, identificaram 2 domínios regulatórios cis-atuantes, denominados CRD1 (-358 a -226) e CRD2 (-226 a -82). A região CRD1 exibe atividade de enhancer, já que foi capaz de restaurar altos níveis de expressão basal do gene repórter gus, quando ligada à extremidade 5’ na posição -82 do promotor de BiP6. A região CRD2 contém tanto cis -elementos regulatórios positivos quanto cis -elementos negativos que contribuem para o padrão tecido-específico de expressão controlado pelo promotor de gsBiP6. A expressão do gene repórter na região do procâmbio em raízes, no meristema apical e no floema de caule foi absolutamente dependente da região CRD2. Em contraste, deleção de CRD2 resultou em acúmulo acentuado de atividade de GUS no parênquima xilemático. A ativação dos promotores BiP6 e BiP9 em resposta ao acúmulo de proteínas anormais no retículo endoplasmático (UPR) e em respostas a estresse osmótico foi avaliada em discos foliares transgênicos. Tratamento dos discos foliares com tunicamicina, ativador da via UPR, e com PEG, indutor de um estresse osmótico, induziu a expressão de GUS, sob o controle do pro motor BiP6 e do promotor BiP9. A deleção da região CRD2 (-226 a -82) no promotor BiP6 causou a perda da inducibilidade da expressão gênica em resposta a tunicamicina e PEG, indicando que CRD2 contém elementos cis-atuantes de resposta a estresse. De fato, dois cis-elementos conservados atuantes na via UPR (UPRE) foram identificados na região CRD2. Coletivamente, estes resultados sugerem que o controle da expressão de BiP em plantas depende de uma complexa integração de múltiplos cis-elementos regulatórios no promotor.
The binding protein BiP is involved in the correct folding of secretory proteins and retention in the RE of unfolded proteins. BiP also prevents nonproductive intermolecular interactions of folding intermediates and subsequent misaggregation of proteins during stress conditions. We have isolated two genomic clones named gsBiP6 and gsBiP9 from λEMBL3 and λZAP II genomic libraries, respectively. The gsBiP6 and gsBiP9 promoters have general motifs founded in plant gene promoters, such as CAAT and TATA sequence, and specific motifs, such as endoplasmatic reticulum stress response element (ERSE) and G-box elements. The BiP promoters were fused to the reporter gene β-glucuronidase (GUS) and introduced into tobacco plants via Agrobacterium tumefaciens transformation. Histochemical analysis of transformed plants with the constructions -358pbip6-gus or -2200pbip9-gus revealed that the BiP promoters produced a uniform pattern of the GUS expression in leaves, stems, and roots. Intense GUS histochemical activity was observed in the vascular region of leaves,stems and roots, and in the leaf trace region as well as in the apical meristem, place where intense cellular division happens. This pattern of space distribution of the GUS activity suggests to be correlated with an expression of those genes in places that have high cellular secretory activity and high proportion of cells in an active process of cellular division. Coherent with this observation, the histochemical activity of GUS is much less intense in the xylem parenchyma. The gsBiP6 promoter deletions were driven in the 5'-3' orientation (-138pbip6-gus) and intern region Δ(-226/- 82)pbip6-gus. Analysis of those deletions in transformed plants identified two regulatory domains cis-acting, denominated CRD1 (-358 to -226) and CRD2 (- 226 to -82). The CRD1 exhibits enhancer activity, since it was capable to recuperate high levels of basal expression of the gus reporte r gene when linked to the extremity 5' in the position -82 of the BiP6 promoter. The CRD2 contains positive regulatory cis -elements, as negative regulatory cis -elements that contribute to the specific pattern of expression controlled by the gsBiP6 promoter. The reporter gene expression in the root procambium region, in the apical meristem and in the phloem stem, was absolutely dependent on the CRD2. In contrast, CRD2 deleted resulted in accentuated accumulation of GUS activity in the xylem parenchyma. The activation of the BiP6 and BiP9 promoters, in response to the unfolded proteins response in the endoplasmatic reticulum (UPR) and osmotic stress condition, were evaluated in transgenic leaf disks. Leaf disks incubated with tunicamycin, activator of the UPR, and with PEG, inductor of an osmotic stress, induced the expression of GUS, under the BiP6 and BiP9 promoters control. The CRD2 (-226 to -82) deleted in the BiP6 promoter caused the loss of the induction of the expression in response to tunicamycin and PEG, indicating that CRD2 contains responsive cis-acting elements under stress conditions. In fact, two conserved cis-acting elements in the UPR (UPRE) were identified in the CRD2. Collectively, these results suggest that the control of the expression of BiP in plants depends on a complex integration of multiple regulatory cis-elements in the promoter.
The binding protein BiP is involved in the correct folding of secretory proteins and retention in the RE of unfolded proteins. BiP also prevents nonproductive intermolecular interactions of folding intermediates and subsequent misaggregation of proteins during stress conditions. We have isolated two genomic clones named gsBiP6 and gsBiP9 from λEMBL3 and λZAP II genomic libraries, respectively. The gsBiP6 and gsBiP9 promoters have general motifs founded in plant gene promoters, such as CAAT and TATA sequence, and specific motifs, such as endoplasmatic reticulum stress response element (ERSE) and G-box elements. The BiP promoters were fused to the reporter gene β-glucuronidase (GUS) and introduced into tobacco plants via Agrobacterium tumefaciens transformation. Histochemical analysis of transformed plants with the constructions -358pbip6-gus or -2200pbip9-gus revealed that the BiP promoters produced a uniform pattern of the GUS expression in leaves, stems, and roots. Intense GUS histochemical activity was observed in the vascular region of leaves,stems and roots, and in the leaf trace region as well as in the apical meristem, place where intense cellular division happens. This pattern of space distribution of the GUS activity suggests to be correlated with an expression of those genes in places that have high cellular secretory activity and high proportion of cells in an active process of cellular division. Coherent with this observation, the histochemical activity of GUS is much less intense in the xylem parenchyma. The gsBiP6 promoter deletions were driven in the 5'-3' orientation (-138pbip6-gus) and intern region Δ(-226/- 82)pbip6-gus. Analysis of those deletions in transformed plants identified two regulatory domains cis-acting, denominated CRD1 (-358 to -226) and CRD2 (- 226 to -82). The CRD1 exhibits enhancer activity, since it was capable to recuperate high levels of basal expression of the gus reporte r gene when linked to the extremity 5' in the position -82 of the BiP6 promoter. The CRD2 contains positive regulatory cis -elements, as negative regulatory cis -elements that contribute to the specific pattern of expression controlled by the gsBiP6 promoter. The reporter gene expression in the root procambium region, in the apical meristem and in the phloem stem, was absolutely dependent on the CRD2. In contrast, CRD2 deleted resulted in accentuated accumulation of GUS activity in the xylem parenchyma. The activation of the BiP6 and BiP9 promoters, in response to the unfolded proteins response in the endoplasmatic reticulum (UPR) and osmotic stress condition, were evaluated in transgenic leaf disks. Leaf disks incubated with tunicamycin, activator of the UPR, and with PEG, inductor of an osmotic stress, induced the expression of GUS, under the BiP6 and BiP9 promoters control. The CRD2 (-226 to -82) deleted in the BiP6 promoter caused the loss of the induction of the expression in response to tunicamycin and PEG, indicating that CRD2 contains responsive cis-acting elements under stress conditions. In fact, two conserved cis-acting elements in the UPR (UPRE) were identified in the CRD2. Collectively, these results suggest that the control of the expression of BiP in plants depends on a complex integration of multiple regulatory cis-elements in the promoter.
Descrição
Palavras-chave
Proteína BiP, Soja, Análise Funcional
Citação
BUZELI, Reginaldo Aparecido Alves. Caracterização molecular de genes que codificam a proteína BiP de soja e análise funcional de seus promotores. 2001. 89 f. Dissertação (Mestrado em Fisiologia Vegetal) - Universidade Federal de Viçosa, Viçosa. 2001.