RESUMO
The expression pattern of tissue-specific promoters in transgenes can be influenced by promoter/enhancer elements employed for the expression of selectable marker genes or elements found in DNA flanking the insertion site. We have developed an analytical system in Arabidopsis thaliana to investigate strategies useful in blocking or reducing nonspecific interactions. These experiments confirm that the DNA configuration and the insertion of spacer DNA aid in the appropriate expression of tissue-specific promoters. It is also demonstrated that the novel tobacco cryptic promoter (tCUP), when used to replace the cauliflower mosaic virus (CaMV) 35S promoter/enhancer, does not show nonspecific interactions. Furthermore, it is shown that insulators isolated from yeast and animals may have potential application in plants. Our results may allow the design of strategies that, individually or in combination, can be used to minimize nonspecific interactions and to design vectors for individual tissue-specific promoters.
Assuntos
Arabidopsis/genética , Regiões Promotoras Genéticas , Transgenes , Caulimovirus/genética , DNA Bacteriano/genética , DNA de Plantas/genética , DNA Espaçador Ribossômico/genética , Regulação da Expressão Gênica de Plantas , Genes Reporter , Marcadores Genéticos , Plantas Geneticamente Modificadas/genética , Plasmídeos , Transformação GenéticaRESUMO
Wild-type tobacco (Nicotiana tabacum L.) seed development was characterized with respect to architecture and carbohydrate metabolism. Tobacco seeds accumulate oil and protein in the embryo, cellular endosperm and inner layer of the seed coat. They have high cell wall invertase (INV) and hexoses in early development which is typical of seeds. INV and the ratio of hexose to sucrose decline during development, switching from high hex to high suc, but not until most oil and all protein accumulation has occurred. The oil synthesis which coincides with the switch is mostly within the embryo. INV activity is greater than sucrose synthase activity throughout development, and both activities exceed the demand for carbohydrate for dry matter accumulation. To investigate the role of INV-mediated suc metabolism in oilseeds, genes for yeast INV and/or hexokinase (HK) were expressed under a seed-specific napin promoter, targeting activity to the apoplast and cytosol, respectively. Manipulating the INV pathway in an oilseed could either increase oil accumulation and sink strength, or disrupt carbohydrate metabolism, possibly through sugar-sensing, and decrease the storage function. Neither effect was found: transgenics with INV and/or HK increased 30-fold and 10-fold above wild-type levels had normal seed size and composition. This contrasted with dramatic effects on sugar contents in the INV lines.
Assuntos
Hexoses/metabolismo , Sementes/crescimento & desenvolvimento , Sacarose/metabolismo , beta-Frutofuranosidase/genética , Metabolismo dos Carboidratos , DNA Bacteriano/genética , Vetores Genéticos , Glucosiltransferases/metabolismo , Hexoquinase/metabolismo , Fosfofrutoquinases/metabolismo , beta-Frutofuranosidase/metabolismoRESUMO
The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.
Assuntos
Brassica napus/genética , Proteínas de Plantas/genética , Sementes/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Arabidopsis/embriologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassica napus/embriologia , Brassica napus/crescimento & desenvolvimento , Divisão Celular , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismoRESUMO
We describe a gene, SCB1 (Seed Coat BURP-domain protein 1), that is expressed specifically within the soybean (Glycine max [L.] Merrill) seed coat early in its development. Northern blot analysis and mRNA in situ hybridization revealed novel patterns of gene expression during seed development. SCB1 mRNA accumulated first within the developing thick-walled parenchyma cells of the inner integument and later in the thick- and thin-walled parenchyma cells of the outer integument. This occurred prior to the period of seed coat maturation and seed filling and before either of the layers started to degrade. SCB1 may therefore play a role in the differentiation of the seed coat parenchyma cells. In addition, the protein product appears to be located within cell walls. The SCB1 gene codes for a new member of a class of modular proteins that possess a carboxy-terminal BURP domain and a variety of different repeated sequences. The sequence of the genomic clone revealed the insertion of a Tgm transposable element in the upstream promoter region but it is not certain whether it contributes to the tissue-specific pattern of SCB1 expression.
