P. Delange et al., CONDITIONAL INHIBITION OF BETA-GLUCURONIDASE EXPRESSION BY ANTISENSE GENE FRAGMENTS IN PETUNIA PROTOPLASTS, Plant molecular biology, 23(1), 1993, pp. 45-55
Antisense RNA-mediated inhibition of gene expression is a valuable too
l to induce mutant phenotypes. We are interested in the application of
antisense gene fragments with the aim to improve the efficiency of in
hibition and to be able to selectively suppress gene family members in
plants. Protoplasts may provide a rapid system to screen the efficien
cy of antisense gene segments. As a first step, we set up a transient
expression system for leaf protoplasts of Petunia hybrida and used as
a model system the inhibition of beta-glucuronidase (uidA) expression
by uidA antisense gene segments. Both GUS enzyme activities and uidA R
NA levels were measured. Co-introducing equal amounts of a full-length
uidA antisense gene and a uidA sense gene reduced GUS activity by 60-
70%. Various uidA antisense fragments also inhibited expression althou
gh with different efficiencies and we show that strong antisense fragm
ents can be retrieved from weak antisense gene fragments. A promoter-l
ess antisense gene did not reduce uidA expression indicating that the
inhibition is mediated by antisense transcripts. Using quantitative PC
R on first-strand cDNA we show that expression of functional antisense
genes lead to reduced levels of uidA mRNA. This suggests that the mec
hanism of antisense RNA inhibition in protoplasts is similar to that i
n transgenic plants and that the protoplast system in combination with
PCR can be used to preselect antisense fragments of any gene.