Construct design for efficient, effective and high-throughput gene silencing in plants
2001
E00050
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Title
Construct design for efficient, effective and high-throughput gene silencing in plants
Author
Wesley, S.V.
Helliwell, C.A.
Smith, N.A.
Wang, M.B.
Rouse, D.T.
Liu, Q.
Gooding, P.S.
Singh, S.P.
Abbott, D.
Stoutjesdijk, P.A.
Robinson, S.P.
Gleave, A.P.
Green, A.G.
Waterhouse, P.M.
Helliwell, C.A.
Smith, N.A.
Wang, M.B.
Rouse, D.T.
Liu, Q.
Gooding, P.S.
Singh, S.P.
Abbott, D.
Stoutjesdijk, P.A.
Robinson, S.P.
Gleave, A.P.
Green, A.G.
Waterhouse, P.M.
Publication Date
2001
Call Number
E00050
Summary
Posttranscriptional silencing of plant genes using antisense or cosuppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study, we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants (including Nicotiana tabacum cv. W38, cotton, rice and Arabidopsis thaliana) showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/antisense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater that that obtained using either cosuppression or antisense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of 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 system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
Journal Citation
v.27(6):581-590, PLANT JOURNAL
Contact Information
harvest@worldveg.org
Record Appears in
Research > Published Articles