Isolation And Identification Of Virus DsRNA From Strawberry Leaves And Analysis Of Their Partial Genome Sequences

Strawberries are perennial and herbaceous plants propagated generally by stolons, which are seriously infected by viruses resulting in expensive impact on strawberry production. So it is important to study the method of virus isolation and the analysis of genome sequences. The aim of this study is to develop a method of isolation and identification of virus dsRNA and elucidate the nucleotide sequences of strawberry viruses. The main results are as follows:1. By using sap inoculation technique, strawberry viruses were successfully transmitted from F.vesca to C.quinoa and showed evident spot symptom. It was proved infecting SMoV and SMYEV by RT-PCR.2. Comparing different methods of extracting dsRNA, the results showed: phenol of pH4.5 easily made the strawberry tissues degradation and influenced the extracted effect, but phenol of pH8.0 could effectively keep the nucleic acid alive. With conventional method of SDS, the extracted virus dsRNA was a fat lot and difficult to study the effects of DNase I, RNase A and two times passing CF-11. The extracted virus dsRNA was so bad that the bands showed dispersion with the method of CTAB. With the modified method of this study, virus dsRNA was successfully extracted from strawberry virus indicator plants and cultivated strawberry plants, and detected through employing agarose gel electrophoresis stained with EB and sequenced.3. The detected effect of agarose gel stained by SYBR was at least higher two double than that stained by EB, but the bands showed evident shift and distortion. The size of virus dsRNA couldn't be correctly shown.4. Only a band of 2kb was extracted from the leaves of infected C.quinoa, but the viruses dsRNA directly extracted from strawberry plants were 5-7kb.5. Through extracting virus dsRNA from different cultivar, cultivation and maturation degree of leaves, the result showed that the quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from leaves of in vitro plantlets was higher than that from the young leaves of field plants. For the field-grown plants, there was more dsRNA in the young leaves.6. In order to obtain dsRNA without DNA and ssRNA, DNase I and RNase A were used. The results from different treated time and concentration indicated that the dsRNA was resistant to DNase I in the presence of 0-0.7 M NaCl, but it became evidently resistant to RNase A only in the presence of 0.5 M NaCl. The plausible way found in the study was to treat the samples by DNase I in 0 M NaCl for 120 min, after depositing of absolute...