Assessment of ribozyme-mediated gene suppression using a luciferase reporter gene containing the sequence of Chrysanthemum Stunt Viroid as a target

作者：Ando Yuko; Oda Kenji; Iwabuchi Masaki; Mino Masanobu^*

来源：Molecular Breeding, 2006, 18(3): 209-218.

DOI：10.1007/s11032-006-9028-z

• Chrysanthemum Stunt Viroid
• hammerhead ribozyme
• particle bombardment
• Luc reporter gene
• in vivo assay

Transgenic tobacco plants expressing hammerhead ribozyme (Rz), which specifically cleaves Chrysanthemum Stunt Viroid (CSVd) RNA, were produced via Agrobacterium-mediated transformation. To analyze the effects of Rz in cells, fire-fly luciferase (Luc) reporter genes, driven by the Cauliflower Mosaic Virus 35S promoter, which have the entire sense or anti-sense cDNA of the CSVd genome in between the Luc ORF and nopalin synthase terminator were constructed. Using particle bombardment, the Luc reporter constructs were delivered into the leaves of the transgenic tobacco plants. Levels of Luc mRNA and Luc enzyme activity were lower in the transgenic plants than in control wild-type plants, suggesting that the transcript of the Luc reporter gene was made unstable in the presence of Rz. On the other hand, mutations at the cleavage sites of the CSVd sequence in the reporter constructs suppressed the reduction of Luc enzyme activity in transgenic plants. A possibility of post-transcriptional gene silencing was ruled out. A direct evidence of the cleavage activity in cells by the experiment of rapid amplification of 5' cDNA end could not obtained, but the present results suggest that the levels of Luc mRNA and Luc enzyme activity was affected by the ribozymes.

• 出版日期2006-10