High-throughput vectors for efficient gene silencing in plants

Helliwell, Chris A., Varsha Wesley, S., Wielopolska, Anna J., & Waterhouse, Peter M. (2002) High-throughput vectors for efficient gene silencing in plants. Functional Plant Biology, 29(10), pp. 1217-1225.

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Abstract

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs, pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

Impact and interest:

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108 citations in Web of Science®

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ID Code: 65976
Item Type: Journal Article
Refereed: Yes
Additional URLs:
Keywords: Genes, Mutagenesis, Polymers, RNA, Phenotype, Plants (botany), gene, Arabidopsis, Embryophyta, Eukaryota
DOI: 10.1071/FP02033
ISSN: 1445-4408
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 2002 C S I R O Publishing
Deposited On: 09 Jan 2014 07:18
Last Modified: 09 Jan 2014 07:19

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