Development of an efficient cis ‐ trans ‐ cis ribozyme cassette to inactivate plant genes

Summary Inactivation of a targeted gene is one of the main strategies used to understand their precise cellular role. In plants, apart from chemical or physical mutagenesis and random insertions of DNA elements followed by screening for a desired phenotype, the most common strategy to inhibit the expression of a given gene involves RNA silencing. This can be achieved either through antisense suppression, sense over‐expression leading to co‐suppression, or expression of double‐stranded DNA constructs (dsRNA). The use of ribozymes to inhibit gene product accumulation has only been occasionally attempted, mainly because of the more complex genetic engineering procedure involved, although the specificity of ribozymes can be an important factor when targeting close members of a gene family. We report here the development of a new cis ‐acting ribozyme cassette for the production of RNAs with desired termini. Attention to many details has been brought in order to provide a powerful procedure for plant application. For example, ultrastable GNRA tetraloops were substituted for both loops II and III of cis ‐acting hammerhead sequences, thereby favouring folding into the catalytically active structure that results in the self‐cleavage of all transcripts. We demonstrate the usefulness of this cassette by producing a ribozyme that cleaves in trans , originally embedded in the cis ‐acting self‐cleaving cassette. The activity of the cis‐trans‐cis construct, was demonstrated both in vitro and in vivo , in transgenic plants with the specific cleavage of an mRNA encoding a 2‐oxo‐glutarate‐dependant dioxygenase predominantly expressed in pistils tissues and in leaves, from the wild potato Solanum chacoense .