ISHS Acta Horticulturae 235: XIV International Symposium on Fruit Tree Virus Diseases
CONSTRUCTION OF MOLECULAR CLONES FOR IDENTIFICATION AND DETECTION OF TOMATO RINGSPOT AND ARABIS MOSAIC VIRUSES |
| Authors: | A. Hadidi, R.W. Hammond |
| Abstract:
Cloning experiments were carried out with an isolate of tomato ringspot virus associated with both Prunus stem pitting disease of stone fruits and apple union necrosis and decline disease in the United States. The arabis mosaic virus isolate associated with hop nettlehead disease in the United Kingdom was also cloned. Viral genomic RNAs were used as templates for cDNA synthesis using random sequence oligonucleotide primers and cloned Moloney-murine leukemia virus reverse transcriptase. E. coli DNA polymerase I and RNase H were then used to produce ds cDNA. Poly dC-homopolymeric tails were added to the 3' ends of the ds DNA with terminal deoxynucleotidyl transferase. These molecules were then annealed to oligo dG-tailed Pst I-digested pBR322 and used to transform competent cells of E. coli HB101. Plasmids with inserts specific for each viral genome were identified by Northern blot and Southern blot hybridization analyses. Selected inserts were subcloned into the polylinker region of the SP6 transcription vector pSP64. Using the SP6 promoter flanking this region, high specific activity cRNA probes were generated by SP6 RNA polymerase. Each probe is specific and can be used for viral identification and detection of infected plants. | |
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