ISHS Acta Horticulturae 130: XII International Symposium on Fruit Tree Virus Diseases
A NUCLEIC ACID ASSOCIATED WITH NECROTIC RUSTY MOTTLE-DISEASED CHERRIES |
| Authors: | D.L. Moore, H.R. Cameron |
| Abstract:
Using a procedure which is highly selective for double-stranded RNA (ds RNA), a nucleic acid species has been isolated from vascular tissue of sweet cherries exhibiting symptoms of Necrotic Rusty Mottle (NRM). Fifteen grams of bark tissue stripped from twigs were crushed in liquid nitrogen. The tissue was thawed in 30 ml GPS buffer (0.2M glycine, 0.1M Na2HPO4, 0.6M NaCl, pH 9.5) containing 0.3g SDS, 0.3g sodium diethyldithiocarbamate (DIECA), 0.75g insoluble polyvinylpyrrolidone (PVP), 0.15g magnesium acetate, plus 30 ml chloroform and 30 ml water-saturated phenol, containing 0.1% 8-hydroxyquinoline. The mixture was centrifuged at 7000g for 15 min. at 4°C. The aqueous phase was recovered and extracted with non-ionic cellulose (Cellex N-l) in the presence of 15% ethanol. The cellulose was collected by centrifugation and washed with STE buffer (0.1M NaCl, 0.5M Tris, 0.001M EDTA, pH 7.0) containing 15% ethanol (15% STE). The nucleic acid was eluted with STE buffer, re-extracted with Cellex N-l and washed again with 15% STE. After elution with STE buffer, the nucleic acid was stored in 66% ethanol for 1.5 hours at -15°C. The resulting preparation was separated by electrophoresis in polyacrylamide gels and stained with "Stains-all". A major band, estimated to be slightly larger than 2 x 106 d., and a minor band, of approximately 1.5 x 106 d. were observed. Brome mosaic virus ds RNA (2.2, 1.98, 1.50, 0.56 x 106 d.) was used as a standard for size comparison. | |
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