RIBOZYME MEDIATED TARGETING OF CUCUMBER MOSAIC-VIRUS RNA-1 AND RNA-2 IN TRANSGENIC TOBACCO PLANTS

Hammerhead ribozymes have been extensively used to inhibit the express ion of cellular genes or viral genes mainly in the animal study, In th is study, we designed a ribozyme targeting the conserved leader sequen ces of cucumber mosaic virus (CMV) RNA 1 and 2. The ribozyme, with asy mmetric lengths of flanking complementary regions, cleaved a model sub strate RNA efficiently at 26 degrees C as well as at 37 degrees C or 5 0 degrees C in vitro. And the ribozyme encoding sequence was introduce d into tobacco plants and expressed with the CaMV 35S promoter and 3' NOS terminator in a monomeric type (pBIR1), tandemly repeated type (pB IR3), and cotranscriptionally combined type (pRokR) with 2.2 copies of I17N satellite RNA, Virus challenging experiments in F-1 plants of re spective transformants with CMV-Y showed specific reductions of viral RNA 1 and 2 in comparison with RNA 3 or 4. Although young plants of a three-leaf-stage showed rather similar mild symptom attenuations in al l constructions compared to CMV-Y inoculated wild type, fully grown pl ants showed a differential degree of resistance upon systemic infectio ns of CMV-Y in pRokR, pBIR3 and pBIR1 transformed plants in a decreasi ng order.