Development of a heat shock inducible expression cassette for plants: characterization of parameters for its use in transient expression assays.
A heat-inducible expression cassette has been constructed to study the conditional expression of sense or antisense orientations of any sequence of interest in transgenic plants or plant tissues. The construct includes the promoter and all but 5 bases of the mRNA leader from the soybean Gmhsp17.5-E gene, the polylinker from pUC18 (modified to remove the ATG), and a fragment that contains the polyadenylation signal and site from the nopaline synthase gene. Analysis of transient expression of a construct containing the beta-glucuronidase (GUS) coding sequence cloned in the cassette and introduced into Nicotiana plumbaginifolia protoplasts by electroporation shows that the promoter has high expression at heat shock temperatures. This construct is expressed at a roughly 80-fold higher level per unit time than a cauliflower mosaic virus 35S gene promoter-GUS construction. The heat shock promoter is regulated positively by supercoiling in this transient assay system. The level of expression of HS-GUS constructions with the polyadenylation sites from either the nopaline synthase gene or the Gmhsp17.5-E gene was similar. Constructs with a perfect fusion at the 5' end had higher levels of expression than those with the corresponding nonperfect transcriptional fusion.
Ainley
W M
WM
Botany Department, University of Georgia, Athens.
Key
J L
JL
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering; D005966:Glucuronidase / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics*; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D013025:Glycine max / Q000235:genetics; D014026:Nicotiana / Q000235:genetics
D006360:Heat-Shock Proteins; D004247:DNA; D005966:Glucuronidase