The analysis of virus genome is based on nucleic acid isolation. The aims of this study were to develop a method for isolation and identification of virus double-stranded ribonucleic acid （dsRNA） and to elucidate the nucleotide sequences of strawberry virus. Using the modified method, virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants and detected using agarose gel electrophoresis with ethidium bromide staining and reverse transcription-polymerase chain reaction （RT-PCR）. The quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from 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. Virus dsRNA extracted from strawberry plants was resistant to deoxyribonuclease Ⅰ （DNase Ⅰ）, but evidently, it became resistant to ribonuclease A （RNase A） only in the presence of 0.5 M NaCl. Its bands in agarose gel could be readily recycled using an agarose gel DNA purification kit. With RT-PCR, the segments of both strawberry mottle virus and Strawberry mild yellow edge virus genomes were amplified by using the virus dsRNA recycled from gel or treated with DNase Ⅰ /RNase A as templates. The system developed for dsRNA isolation and identification in strawberry plants laid a sound foundation for the work on genome analysis of strawberry virus isolates in China.
LI He DAI Hong-yan ZHANG Zhi-hong GAO Xiu-yan DU Guo-dong ZHANG Xin-yu
The 930 bp segment in 3’ terminal region of Strawberry mild yellow edge virus(SMYEV)genome was amplified by 3’ rapid amplification of cDNA ends(RACE).Ten Chinese isolates were sequenced,and 7 of them were the same.Nucleotide and amino acid identities and phylogenesis were analyzed between Chinese isolates and 24 isolates from other regions of the world.Sequence analysis of the 878 nt stretch within 3’ terminal region of SMYEV genome showed that nucleotide acid identities ranged from 79.5% to 100%,deduced amino acid sequences of coat protein gene identity were 86.4% to 100%.Phylogenetic analysis showed that all isolates of SMYEV fell into four clades.To a certain extent,the clades were related with the geological distribution of SMYEV.Chinese isolates SY01 and SY04 lay in the same clade with European and American isolates,but formed a small separate branch.Isolates SY03 and SY02,derived from Fragaria×ananassa cv.Changhong-2 and F.pentaphylla respectively,had a far relationship with other isolates and fell into one clade.They were likely to be the special isolates that existed only in China.
Coat protein gene of 12 isolates of Strawberry vein banding virus(SVBV) was studied by multiple sequence alignment and the primers located in conserved region were designed.The detection protocol for SVBV by reverse transcriptase polymerase chain reaction(RT-PCR) was developed.The primers of multiplex RT-PCR were selected by primer-primer interactions and the melting temperature.The annealing temperature,the concentration of PCR buffer,the extension temperature,the extension time and the concentration of pri-mers were optimized,respectively.A multiplex RT-PCR assay was made for simultaneous detecting Strawberry mottle virus(SMoV),Strawberry mild yellow edge virus(SMYEV) and SVBV.Both field-grown strawberries and microplants were detected effectively.It was the first report that multiplex RT-PCR was used to assay the efficacy of strawberry viruses elimination.
