Cloning and expression in E. coli of grapevine leaf-roll associated virus-3 RdRp gene

The dsRNA was extracted from scraped phloem tissue of grapevine canes infected by grapevine leafroll-associated virus-3(GLRaV-3) and used as template for cDNA synthesis. The RNA-dependent RNA polymerase (RdRp) gene of GLRaV-3 was cloned and sequenced. The complete gene consists of 1618 nucleotides and encodes a polypeptide consisting of 538 amino acids(aa). Comparison of the sequences with those of the isolate NY1 of GLRaV-3 showed 99.3% similarity in nucleotide sequence and 99.6% similarity in amino acid sequence. Alignment of the polypeptide with RdRp of pineapple mealybug wilt associated virus-2(PMWaV-2), grapevine leafroll associated virus-1(GLRaV-1) and little cherry virus-2 (LCV-2) which were in the same genus with GLRaV-3 revealed the presence of eight conserved motifs including GDD. The similarities between RdRp of GLRaV-3 and that of PMWaV-2, GLRaV-1 and LCV-2 were 53%, 50% and 38%, respectively. The recombinant plasmid pET22b containing RdRp was transformed into E. coli BL21 (DE3) plysS. Results of SDS-PAGE showed that specific expression of a 61 kDa fusion protein was achieved by the inducing of 1mmol/L IPTG.