ホップ矮化ウイロイドの精製

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  • Purification of Hop Stunt Viroid
  • ホップ ワイカ ウイロイド ノ セイセイ エイブン

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Purification of hop stunt viroid (HSV) from the infected cucumber leaf tissues has been accomplished. Comparative analysis of three extraction methods for low molecular weight RNA as well as HSV, that is, methods of Diener et al., Sänger et al. and Niblett et al., indicates no discernible differences in the yields of extractable RNA (30-32mg/kg of infected tissues). In this work, a more rapid extraction procedure was developed by modifying the direct phenol method of Diener et al. HSV was then purified to homogeneity from this extract by two-cycles of gel electrophoresis. Specific HSV band was consistently detected on 7.5% polyacrylamide gel electrophoresis. The yield of purified HSV was 30-40μg/kg of infected tissues. The dilution end point for infection of the purified HSV was between 10 and 100pg per ml, and its 50% infective dose (ID50) was 5ng per ml. The electrophoretic mobility of non-denatured HSV was markedly affected by acrylamide concentrations; apparent molecular weight of HSV was estimated at 52, 000-69, 000 daltons, whereas molecular weight estimate of 96, 000 daltons was derived from determination under denaturing condition. Denatured HSV was separated into two electrophoretic bands on polyacrylamide gels (5, 7.5 and 10%) containing 8M urea at 60C. Both slower migrating and faster migrating bands were infectious when bioassayed on cucumber plants.

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