title: Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase.
abstract: Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.
authors.affiliation: Calgene Inc., Davis, CA 95616.
authors.forename: K E
authors.initials: KE
authors.lastname: McBride
authors.forename: D J
authors.initials: DJ
authors.lastname: Schaaf
authors.forename: M
authors.initials: M
authors.lastname: Daley
authors.forename: D M
authors.initials: DM
authors.lastname: Stalker
mesh_terms: D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017343:Genes, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D018087:Plastids / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics*; D014764:Viral Proteins
chemical_list: D012333:RNA, Messenger; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D005966:Glucuronidase