RNA interference (RNAi) is a phenomenon in which small double-stranded RNA molecules induce sequence-specific degradation or inhibit the translation of homologous single-stranded RNA. In plants, RNAi can be induced through transfection or microinjection of long double-stranded RNA. The double-stranded RNA is cleaved into short RNA fragments of about 19 to 23 nucleotides (siRNAs). siRNAs are incorporated into a ribonuclease enzyme complex known as the RNA-induced silencing complex (RISC). The antisense strand of siRNA within the RISC pathway serves as a guide for sequence-specific degradation of homologous messenger RNAs.
The ability of transfected synthetic small interfering RNAs to suppress the expression of specific transcripts has proven to be a useful tool to study gene function. Recently short hairpin RNAs (shRNAs) have been shown to silence genes as effectively as short dsRNAs. Several DNA-based vectors have been developed that direct transcription of small hairpin RNAs (shRNAs). These RNAs are processed into functional siRNAs by cellular enzymes. RNAi vectors for the expression of shRNAs are available. These vectors typically use RNA polymerase III (Pol III) to express short hairpin RNAs. These transcripts adopt stem-loop structures that are processed into siRNAs by the RNAi machinery.
Other vectors have been developed that drive expression of both the sense and antisense strands of a DNA construct separately. The transcripts hybridize in vivo to make the siRNA. In efforts to induce long-term gene silencing, expression vectors that continually express siRNAs in stably transfected cells have been used.
One of the limitations in using hairpin technology for gene silencing is that silencing of many genes can be lethal or detrimental to recovering plants and/or progeny. In addition, another common problem is that assembly of hairpin DNA constructs in cloning vectors using routine molecular biology methods can often be difficult due to instability of such constructs in bacterial hosts used for cloning. Methods and compositions are needed in the art which improved the manner in which gene silencing is performed in plants.
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