Nucleotide Sequence Of DsRNA Virus Infecting Pleurotus Ostreatus And Virus Eliminate Method

In 2006, we isolated virus from the mycelium pellet of Tianda-300,Pleurotus ostreatus,but the number of viral type are not easy to determine. Further,we achieved the purified viral particles by PEG sedimeatation, fifferential and sucrosse density gradient centrifugation. Observed by the electron microscope, the purified virus preparations contained isometric particles of 21～23nm in diameter. The SDS-PAGE electrophoretic display it has two kind of molecular weights respectively 39 and the 30kD capsid protein. The genome is 2.3kb dsRNA, the length is 2.3kb.The 2.3kb band extracted from viral particle, but four bands were achieved by the dsRNA technique, the size of the dsRNA were 8.2kb,2.3kb,2.0kb and 1.7kb.So the 8.2kb,2.0kb and 1.7kb bands are naked. Using RT-PCR, the cDNA library construction and the RACE technology,we gained 4083bp,1556bp and 1047bp length cDNA from 8.2kb,2.3kb,2.0kb dsRNA. Through the sequence blast and phylogenetic analysis, the 2.3kb and 2.0kb blongs to partitiviruses and the 8.2kb dsRNA sequence is similar to the viral OMSV,96% similarity. They are all close related to plant virus,and far more close related to mycovirus,and were more distantly related to totiviruses.At the same time, we use four methods to eliminate the virus from mycelia cutting the edge of mycelia colony, the protoplast preparation, the single spore joint and the freeze-drying.Virus-free strains were obtained by single spore joint and protoplast preparation. Virus-free strain can not be obtained by cutting the edge of mycelia colony and the freeze-drying.The two methods only can decrease the dsRNA amount.Virus-free strains displayed excellent characters:the growth speed is quick, the acticity of laccase is high,the myxelia colony is thick sturdy. Among the strains of protoplast preparation, some only have 2.3kb dsRNA band,other have 2.3kb and 2.0kb dsRNAbands.This told us that the exist of 2.3kb dsRNA may not rely on the 2.0kb dsRNA,but the 2.0kb dsRNAg must be rely on in the 2.3kb dsRNA to exist.