Gene Construction and Activity Detection of the Ribozyme Multi-targeting against Negative Strand of Potato Spindle Tuber Viroid in vitro

The genes of hammerhead ribozymes targeting against two and three sites on negative strand of potato spindle tuber viroid (PSTVd) were designed, synthesized and cloned according to the action manner of hammerhead ribozyme. After transcription in vitro , the PSTVd negative RNA was mixed with the ribozyme containing two or three cleavage sites separately and incubated two hours at 37℃. The results showed that the ribozymes containing two and three cleavage sites have highly catalytic activity, and the size of the cleft products and released by the ribozyme containing two cleavage sites is correspond with the expected value. Although the ribozyme containing three cleavage sites has highly catalytic activity, the ribozyme is effective only for one cleavage site. The applied prospect for two ribozymes was discussed in the paper.