Ribozyme mediated targeting of cucumber mosaic virus RNA 1 and 2 in trans genic tobacco

Abstract

Hammerhead ribozymes have been extensively used to inhibit the expression of cellular genes or viral genes mainly in animal study. In this research, we designed a short catalytic RNA targeting the conserved leader sequences of cucumber mosaic virus(CMV) RNA 1 and 2. The ribozyme, with asymmetric lengths of flanking complementary regions, cleaved a model substrate RNA efficiently at 26℃ as well as at 37℃ or 50℃ in vitro. The ribozyme gene was introduced into tobacco plants to be expressed with the context of CaMV 35S promoter and 3 NOS terminator in a monomeric type(BIR1), tandemly repeated type(BIR3), and cotranscriptionally combined type(RokR) with 2.2 copies of $I_{17}N$ satellite RNA. Virus challenge showed specific reductions of viral RNA 1 and 2 in comparison with RNA 3 and 4 in systemic leaves of F1 progenies of plants containing ribozyme gene. Additionally, unit-length satellite RNA was massively produced in systemic leaves showing viral symptoms in F1 generations of RokR plants. Although plants above young stage showed differential resistances to systemic infections of CMV-Y in RokR, BIR3, and BIR1 progenies in a decreasing order, young plants of three leaf stage clearly showed only symptom attenuations in all constructions