| 摘要 |
Methods and constructs for the introduction of multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway are provided. In one embodiment, the constructs contain two or more enzyme-encoding genes, each under the control of an inducible promoter and each with a polyadenylation signal. The constructs are used to produce transgenic plants, in which the expression of the enzymes are increased when a chemical inducing agent is applied, and a biosynthetic product of the series of enzymes encoded by the transgenes is produced. Constructs may be used which contain two or more enzyme-encoding genes under the control of one or more promoters activated by activator molecules or complexes expressed from a transgene or transgenes, which are themselves under the control of one or more inducible promoters and switched on following the external application of a chemical. The transgene or transgenes expressing the activator molecules or complexes may be included in the same construct containing multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway. Alternatively, the transgene or transgenes expressing the activator molecules or complexes may be on a different construct from the construct containing multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway. The activator molecule can be expressed using a constitutive promoter in an inactive form which is converted to the active form following application of the chemical inducing agent. |
| 主权项 |
1. A recombinant plant vector for the expression of enzymes in a biosynthetic pathway comprising three or more elements,
wherein each element comprises operatively linked in the 5′ to 3′ direction: a promoter that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a protein; and a 3′ polyadenylation signal sequence; wherein the promoter is selected from the group consisting of a chemically inducible promoter and a promoter activated by an activator molecule or complex, wherein at least one promoter is a promoter activated by an activator molecule or complex, and at least one promoter is a chemically inducible promoter, wherein said chemically inducible promoter is activated by external application of a chemical, wherein the biosynthetic product is a polyhydroxyalkanoate and the proteins encoded by the nucleic acid sequences are selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, dehydratase, isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes, and wherein inducible expression of the recombinant plant vector in a plant produces at least 10% dwt polyhydroxyalkanoate in leaves of the plant without the plant exhibiting a stunted chlorotic phenotype.
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