Isolamento e caracterização de promotores órgão-específicos a partir de informações do Banco FORESTs (Eucalyptus Genome Sequencing Project Consortium)
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Data
2008-05-26
Autores
Sassaki, Flávio Tetsuo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os cassetes de expressão empregados atualmente para a produção de plantas geneticamente modificadas são baseados, na sua maioria, em promotores constitutivos que determinam a expressão generalizada do transgene na planta, o que muitas vezes não é necessário nem desejado. A alternativa mais viável para substituição de tais promotores é investir na identificação e caracterização de promotores órgão/tecidoespecíficos ou estímulo-dependentes nas espécies de interesse. O presente projeto teve como objetivos identificar e caracterizar genes com padrão de expressão órgãoespecífico em eucalipto e, a partir dessas informações, isolar e caracterizar as seqüências promotoras adjacentes. Predições in silico no banco de dados do projeto de seqüências expressas do eucalipto (FORESTs), e informações disponíveis na literatura a respeito de genes com padrão de expressão órgão-específico, foram utilizadas na seleção. Assim, 59 genes preditos como possuindo expressão exclusiva em dado órgão foram selecionados para a validação via RT-PCR. Dos candidatos validados, 2 eram de raiz, 1 de folha, 1 de botão floral e 1 de botão floral e fruto, concomitantemente. Três candidatos ubíquos também foram selecionados. Dois desses candidatos tiveram seus perfis de expressão em diferentes órgãos de eucalipto avaliados por PCR quantitativa, indicando que o primeiro é preferencialmente expresso em folhas, e o segundo expresso especificamente em raiz. A partir de estratégias de “genome walking” foram isolados diferentes promotores putativos, sendo que o promotor do candidato com expressão preferencial em folha (1.2 kb) foi selecionado para a caracterização funcional em plantas usando o gene uidA, que codifica a -glucuronidase (GUS), como repórter. Em plântulas transgênicas de tabaco da geração T1, a expressão de GUS foi detectada majoritariamente...
Many plant genetic engineering applications require spatial expression of transgenes, which in turn depends upon the availability of tissue- and organ-specific promoters. In the present work, the identification of genes with organ-specific expression in Eucalyptus grandis was performed aiming the subsequent isolation and characterization of contiguous promoter sequences. Candidates genes were selected by in silico predictions in the Eucalyptus EST project (FORESTs) database or by searching the available literature for genes with specific expression patterns. Fifty-nine genes with predicted organ-specific expression were selected for further RT-PCR validation. Among the validated candidate genes, 2 were root-specific, 1 was leaf-specific, 1 was flower-bud-specific and 1 was flower-bud and fruit specific. Three genes ubiquitously expressed were also selected. The relative expression levels of two of them (one leafspecific and one root-specific) over a broad range of eucalyptus organ/tissues were determined using real time PCR. The 5’ putative regulatory sequences of the validated genes were isolated using genome walking strategies, and the activity of the leafspecific promoter (1.2 kb) was further investigated in transgenic tobacco plants using a GUS reporter system. In transgenic seedlings from the T1 generation, GUS expression driven by this promoter was detected preferentially in cotyledons. In adult plants, however, GUS expression was detected in both stem and root, but in lower intensity if compared to the expression observed in leaves. This promoter was also able to modulate the transient expression of GUS in seedlings of Eucalyptus grandis.
Many plant genetic engineering applications require spatial expression of transgenes, which in turn depends upon the availability of tissue- and organ-specific promoters. In the present work, the identification of genes with organ-specific expression in Eucalyptus grandis was performed aiming the subsequent isolation and characterization of contiguous promoter sequences. Candidates genes were selected by in silico predictions in the Eucalyptus EST project (FORESTs) database or by searching the available literature for genes with specific expression patterns. Fifty-nine genes with predicted organ-specific expression were selected for further RT-PCR validation. Among the validated candidate genes, 2 were root-specific, 1 was leaf-specific, 1 was flower-bud-specific and 1 was flower-bud and fruit specific. Three genes ubiquitously expressed were also selected. The relative expression levels of two of them (one leafspecific and one root-specific) over a broad range of eucalyptus organ/tissues were determined using real time PCR. The 5’ putative regulatory sequences of the validated genes were isolated using genome walking strategies, and the activity of the leafspecific promoter (1.2 kb) was further investigated in transgenic tobacco plants using a GUS reporter system. In transgenic seedlings from the T1 generation, GUS expression driven by this promoter was detected preferentially in cotyledons. In adult plants, however, GUS expression was detected in both stem and root, but in lower intensity if compared to the expression observed in leaves. This promoter was also able to modulate the transient expression of GUS in seedlings of Eucalyptus grandis.
Descrição
Palavras-chave
Eucalipto - Melhoramento genético, Genetica vegetal, Expressão gênica, Eucalyptus grandis
Como citar
SASSAKI, Flávio Tetsuo. Isolamento e caracterização de promotores órgão-específicos a partir de informações do Banco FORESTs (Eucalyptus Genome Sequencing Project Consortium). 2008. 104 f. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Botucatu, 2008.