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Plant Chromosome Engineering

Volume 701 of the series Methods in Molecular Biology pp 179-197

Date:

Vectors and Methods for Hairpin RNA and Artificial microRNA-Mediated Gene Silencing in Plants

  • Andrew L. EamensAffiliated withSchool of Microbial and Molecular Sciences, University of Sydney Email author 
  • , Peter M. WaterhouseAffiliated withSchool of Microbial and Molecular Sciences, University of Sydney

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Abstract

In plant cells, DICER-LIKE4 processes perfectly double-stranded RNA (dsRNA) into short interfering (si) RNAs, and DICER-LIKE1 generates micro (mi) RNAs from primary miRNA transcripts (pri-miRNA) that form fold-back structures of imperfectly dsRNA. Both si and miRNAs direct the endogenous endonuclease, ARGONAUTE1 to cleave complementary target single-stranded RNAs and either small RNA (sRNA)-directed pathway can be harnessed to silence genes in plants. A routine way of inducing and directing RNA silencing by siRNAs is to express self-complementary single-stranded hairpin RNA (hpRNA), in which the duplexed region has the same sequence as part of the target gene’s mRNA. Artificial miRNA (amiRNA)-mediated silencing uses an endogenous pri-miRNA, in which the original miRNA/miRNA* sequence has been replaced with a sequence complementary to the new target gene. In this chapter, we describe the plasmid vector systems routinely used by our research group for the generation of either hpRNA-derived siRNAs or amiRNAs.

Key words

dsRNA hpRNA siRNA miRNA amiRNA RNA silencing Plant expression vectors pHellsgate12 and pBlueGreen