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

Wesley, SV Helliwell, CA Smith, NA Wang, MB Rouse, DT Liu, Q Gooding, PS Singh, SP Abbott, D Stoutjesdijk, PA Robinson, SP Gleave, AP Green, AG Waterhouse, PM

Sv. Wesley et al., Construct design for efficient, effective and high-throughput gene silencing in plants, PLANT J, 27(6), 2001, pp. 581-590

31

INGLESE

Article

Plant Sciences","Animal & Plant Sciences

PLANT JOURNAL

0960-7412 → ACNP

27

6

2001

581 - 590

ISI

0960-7412(200109)27:6<581:CDFEEA>2.0.ZU;2-S

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.