Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define an essential gene in methionine biosynthesis

Controlled down-regulation of endogenous plant gene expression is a useful tool, but antisense and sense silencing lack predictability. Recent studies show that expression of both antisense and sense RNA together is an effective means of inactivating reporter and viral genes in plants. We created tr...

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Bibliographic Details
Published in:Plant molecular biology 2000-12, Vol.44 (6), p.759-775
Main Authors: Levin, J Z, de Framond, A J, Tuttle, A, Bauer, M W, Heifetz, P B
Format: Article
Language:English
Subjects:
Arabidopsis
Arabidopsis - drug effects
Arabidopsis - genetics
Arabidopsis - growth & development
Biosynthesis
DNA, Recombinant
Gene expression
Gene Expression Regulation, Plant
Gene Silencing
Genes, Essential - genetics
Genetic Markers
Inactivation
luciferase
Luciferases - genetics
Luciferases - metabolism
Lyases - genetics
Lyases - metabolism
methionine
Methionine - biosynthesis
Methionine - pharmacology
Methods
Phenotype
Plants, Genetically Modified
Plasmids - genetics
RNA
RNA, Double-Stranded - genetics
RNA, Messenger - genetics
RNA, Messenger - metabolism
Transgenic plants
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Summary:Controlled down-regulation of endogenous plant gene expression is a useful tool, but antisense and sense silencing lack predictability. Recent studies show that expression of both antisense and sense RNA together is an effective means of inactivating reporter and viral genes in plants. We created transgenic plants expressing antisense and sense RNA together in a single 'double-stranded RNA' (dsRNA) transcript. This approach shows great promise as a highly effective means for reducing gene function. With this approach, we demonstrated that the Arabidopsis cystathionine beta-lyase gene, which encodes a methionine biosynthetic enzyme, is essential for viability. Inactivation of this gene was rescued by the addition of methionine to the growth medium. Compared to antisense and sense constructs, the dsRNA construct showed a much more consistent and complete suppression of gene activity. Additionally, expression of a transcript with a spacer sequence containing an unrelated gene between antisense and sense luciferase gene fragments led to stronger inactivation of a second luciferase transgene than did constructs with a minimal spacing between sense and antisense fragments. However, the gene in the spacer region was neither functionally expressed nor functional in silencing a second, unlinked homologous transgene.
ISSN:0167-4412
1573-5028
DOI:10.1023/A:1026584607941