ABSTRACT
Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.
Subject(s)
Arabidopsis/genetics , DNA-Binding Proteins/genetics , Ethanol/pharmacology , Fungal Proteins/genetics , Gene Expression Regulation, Plant/drug effects , Regulon , Aspergillus nidulans/genetics , Enzyme Induction , Gene Expression Regulation, Enzymologic/drug effects , Genes, Reporter , Glucuronidase/biosynthesis , Glucuronidase/genetics , Green Fluorescent Proteins , Kinetics , Luciferases/biosynthesis , Luciferases/genetics , Luminescent Proteins/genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Nicotiana/genetics , Transformation, GeneticABSTRACT
Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.
Subject(s)
Alcohol Dehydrogenase/genetics , Carbon/metabolism , DNA-Binding Proteins/genetics , Ethanol/pharmacology , Fungal Proteins/genetics , Gene Expression Regulation, Plant/drug effects , Genetic Vectors , Plants, Genetically Modified , Transcription Factors/genetics , Aspergillus nidulans/genetics , Caulimovirus/genetics , Glycoside Hydrolases/biosynthesis , Phenotype , Photosynthesis , Plants, Toxic , Promoter Regions, Genetic , Regulon , Nicotiana/enzymology , Nicotiana/genetics , Nicotiana/metabolism , Transgenes , beta-FructofuranosidaseABSTRACT
Plasma neurotensin concentrations are rapidly elevated after oral ingestion or intraduodenal infusion of fat, apparently before fat reaches the ileum where neurotensin is highly concentrated. The purpose of this study was to investigate the site of neurotensin release and to determine whether neurotensin is released by direct luminal stimulation by fat in conscious dogs. Dogs were prepared with isolated jejunal or ileal segments and portal vein catheters. Release of neurotensin into the portal venous blood was examined by selective perfusion of each intestinal segment with sodium oleáte. The results of this study show that selective perfusion of the jejunum, but not the ileum, with sodium oleate, caused a significant release of neurotensin. We speculate that release of ileal neurotensin is not due to direct luminal stimulation, but is mediated by local neural or humoral intermediates.
