MOLECULAR CLONING OF DSRNAS ASSOCIATED WITH STRAWBERRY MOTTLE VIRUS

Abstract

Strawberry mottle virus was transmitted from Fragaria vesca to Chenopodium quinoa and Nicotiana occidentalis 37B. In all plants, SMoV infection-associated dsRNA was detected but dsRNA patterns showed some differences between the herbaceous hosts. The differences were more evident when the dsRNA preparations were electrophoresed employing a SDS-PAGE method followed by silver staining of polyacrylamide gels than when employing traditional agarose gel electrophoresis and EtBr staining. Double-stranded RNA, purified from N. occidentalis 37B, was used to synthesize cDNA and for the development a cDNA library in Escherichia coli. Recombinant plasmids were analyzed by restriction enzyme digestion, and specific cDNA clones were selected by means of a 32P-labelled probe prepared from SMoV dsRNA. After a miniprep step, the plasmids were digested and the length of the inserts was determined to be between 200 and 2000 bp. The largest inserts were selected and used for further research. Hybridization experiments confirmed the specificity of the cloned material for SMoV dsRNA.