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

Varsha Wesley, S., Helliwell, Christopher A., Smith, Neil A., Wang, Ming-Bo, Rouse, Dean T., Liu, Qing, Gooding, Paul S., Singh, Surinder P., Abbott, David, Stoutjesdijk, Peter A., Robinson, Simon P., Gleave, Andrew P., Green, Allan G., & Waterhouse, Peter M. (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant Journal, 27(6), pp. 581-590.

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Post-transcriptional silencing of plant genes using anti-sense or co-suppression 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 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 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 than that obtained using either co-suppression or anti-sense 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.

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867 citations in Web of Science®

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ID Code: 65979
Item Type: Journal Article
Refereed: Yes
Additional URLs:
Keywords: Gateway, Genomics, ihpRNA, PTGS, RNAi, Vector, expressed sequence tag, gene expression system, gene silencing, genetic transcription, hairpin RNA, intron, molecular cloning, plant genetics, transgenic plant, Cloning, Enzymes, Genes, RNA, Plants (botany), Arabidopsis, Genetic Vectors, Introns, Molecular Probe Techniques, Molecular Sequence Data, Nucleic Acid Conformation, Oryza sativa, Plants, Genetically Modified, Research Design, RNA Probes, RNA Splicing, RNA, Antisense, Tobacco, Transformation, Genetic
DOI: 10.1046/j.1365-313X.2001.01105.x
ISSN: 0960-7412
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > PLANT BIOLOGY (060700)
Divisions: Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2001 Wiley-Blackwell Publishing Ltd.
Deposited On: 09 Jan 2014 07:34
Last Modified: 13 Jan 2014 00:49

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