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

Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define... 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 β-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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 2000 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1026584607941
Publisher site
See Article on Publisher Site

Abstract

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 β-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.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 16, 2004

References

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