Use of the polymerase chain reaction to clone the potato leafroll virus coat protein gene directly from the total RNA of infected plants

Abstract

The potato leafroll virus (PLRV) coat protein (CP) gene was directly cloned from the total RNA extracted from virus-infected plants. First strand cDNA synthesis was not necessarily specific; it was equally efficient using either random or CP-specific primers. The viral sequence encoding the coat protein was specifically amplified from the total population of cDNA molecules by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was cloned blunt-end into the pT7T318U plasmid vector, and the authenticity of the cloned gene verified by sequence analysis. This cloning strategy obviates the need for virus purification. Sequence comparison of the CP gene of the Italian isolate and those of five other PLRV isolates revealed a close similarity to the three European and the Canadian isolates, and a more distant relationship with the Australian one.