Construct design for efficient, effective and high-throughput gene silencing in plants

Citation
Sv. Wesley et al., Construct design for efficient, effective and high-throughput gene silencing in plants, PLANT J, 27(6), 2001, pp. 581-590
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PLANT JOURNAL
ISSN journal
0960-7412 → ACNP
Volume
27
Issue
6
Year of publication
2001
Pages
581 - 590
Database
ISI
SICI code
0960-7412(200109)27:6<581:CDFEEA>2.0.ZU;2-S
Abstract
Post-transcriptional silencing of plant genes using anti-sense or co-suppre ssion constructs usually results in only a modest proportion of silenced in dividuals. Recent work has demonstrated the potential for constructs encodi ng self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. I n this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti- sense arms ranging from 98 to 853 nt gave efficient silencing in a wide ran ge of plant species, and inclusion of an intron in these constructs had a c onsistently enhancing effect. Intron-containing constructs (ihpRNA) general ly gave 90-100% of independent transgenic plants showing silencing. The deg ree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generi c vector, pHANNIBAL, that allows a simple, single PCR product from a gene o f interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase sys tem. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNA! is being used to examine g ene function in Caenorhabditis elegans.