Abstract: Based on the functional model of the hammerhead RNAs，a divalent ribozyme was designed for cleavage of Potato leafroll virus(PLRV)RNA，targeting at two sites on the negative strand of PLRV Chinese isolate replicase gene and cloned in vector pGEM-4Z． The recombinant plasmid pGEM-4ZDR was useed for in vitro transcription of the ribozyme RNA． The plasmid pGEM-4ZR5 for in vitro transcription of the substrate viral RNA was synthesized by cDNA subcloning of a PLRV Chinese isolate(PLRV-Ch)into plasmid pGEM-4Z，in which an 874 bp cDNA fragment at the 5' end of PLRV-Ch replicase gene was reversely inserted downstream of the T7 promoter in pGEM-4Z． By using T7 DNA polymerase，the plasmids of pGEM-4ZDR and pGEM-4ZR5 were linearized with EcoR I and used as templates for in vitro transcription of the divalent ribozyme or the negative strand RNA of PLRV-Ch replicase gene，respectively． After mixture of the RNA transcripts and incubation at 37℃，results showed that the ribozyme has highly catalytic activity to the PLRV-Ch negative strand RNA in vitro．

Key words: Divalent ribozyme, Potato leaf roll virus(PLRV), Repilcase gene, in vitro cleavage

CLC Number: 

• Q78