Comparative analysis of the efficiencies of siRNA and amiRNA gene silencing of the tobacco lycopene cyclase gene (NsCrtL-1).

摘要

Small interfering RNAs (siRNA) and artificial microRNAs (amiRNA) are widely used for gene silencing in plants, but comparisons of their efficiencies at silencing the same gene have rarely been reported. Both RNAi methods were targeted at the lycopene cyclase gene (NsCrtL-1) in Nicotiana sylvestris. Two amiRNA vectors were constructed, one of which perfectly matched the target gene, while the other contained two nucleotide mismatches. Three vectors (one siRNA vector and two amiRNA vectors) were used to transform tobacco, and the abundances of NsCrtL-1 gene transcripts in the transgenic tobacco plants were determined by real-time reverse-transcription quantitative PCR (RT-qPCR). The results showed that siRNA had the greater silencing efficiency, with target gene transcript levels declining to 9.7% of those in non-transformed, wild-type plants. The gene silencing efficiencies of the two amiRNA vectors were similar. Target gene transcript levels in amiRNA-transgenic plants were reduced to approx. 20% of those in non-transformed, wild-type plants. HPLC was used to measure lycopene concentrations following NsCrtL-1 gene suppression in the three different transgenic plants. The mean lycopene concentration in wild-type control plants was 3.36 micro g g-1 fresh weight (FW), while in siRNA-transgenic plants it increased to 7.37 micro g g-1 FW. In the amiRNA-transgenic plants, mean lycopene concentrations increased to 5.23 micro g g-1 FW and 5.82 micro g g-1 FW for the amiRNA constructs with a perfect or an imperfect target sequence match, respectively. These results indicate a higher efficiency of gene silencing when the siRNA approach was used compared to the amiRNA approach. In the amiRNA constructs, two mismatches between the mature miRNA and the target gene sequence had no significant effect on the efficiency of gene silencing.