Construction Of A Divalent Ribozyme Gene Specific To Potato Leafroll Virus And Its Application For Resistance Of Transgenic Potato

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 replicase gene. By oligonucleotide driven PCR, the recombinant plasmid pGEM-4ZDR was obtained 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 isolate in Chinese (PLRV-Ch) into plasmid pGEM-4Z, in which an 874-bp cDNA fragment at the 5' end of PLRV replicase gene was reversely inserted downstream from the T7 promoter in pGEM-4Z. By using T7 DNA polymerase, the plasmids of pGEM-4ZDR and pGEM-4ZR5 were linearized with EcoRI and used as templates for in vitro transcription of the divalent ribozyme or the negative sense RNAs of PLRV-Ch, 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 RNA in vitro.To obtain virus-resistant transgenic potato lines, a binary vector of plasmids pROKⅡ/DR containing the divalent ribozyme gene was constructed and transferred into Agrobacterium tumefaciens LBA4404 by tri-parental conjugation. The leaf disks of potato cultivar Desiree were used for gene transformation mediated by Agrobacterium. The gene integration in the regenerated progenies was identified by PCR and Southern-blot and results revealed that 14.5% of the regenerated plants were transformed by the ribozyme gene. The gene was existed stably in asexual propagated progeny. Expression of the ribozyme gene on RNA level was also confirmed by both RT-PCR and RNA dot-blots. Detection of the transgenic plants resistant to virus infection by aphid transmission was carried out in the greenhouse and the netroom trials. The transgenic potato lines developed disease phenotypes differently. Among them infected by PLRV, the asexual propagated second progenies of L5, L7, L8 and J-1 line showed relative high levels of resistance. In netroom prevented from aphides, plot test of the transgenic potato lines that were uninfected by the virus showed the different yields, in which the lines of L5 and J-l highly resistant to PLRV were yielded similarly to that of the non-transgene control of potato cultivar Desiree. With these results, it is become possible to develop potato varieties resistant to virus infection by transformation with ribozyme genes.