Establishing And Primary Analyses Of The CDNA Library Of Ramie Mosaic Virus

Ramie mosaic disease is one of the major diseases in ramie. The pathogen of this disease is ramie mosaic virus. Most of the researches of ramie mosaic virus focus on the biological traits interiorly. The researches on classification and genomic structure have not been reported. We classify the ramie mosaic virus to Closteroviruses based on the biological traits primarily. The members of Closteroviruses have the longest genome in plant virus and the relation between the members of them is complex. We establish the cDNA library of ramie mosaic virus and expect to do more research on this virus.In this study, taking the ramie leaves characterized the typical symptom of ramie mosaic disease from Ramie Institution of Hunan Agriculture University as experimental materials;ramie mosaic virus is purified through ultracentrifugation. The baculiform virion is observed under the electron microscope and we can see clearly a long line virus with the size of (1 265±20 nm)×(13±1 nm). After SDS-PAGE analysis of the virus extraction, there is one piece of 25 kDa protein band in gel and we predict it as the coat protein of ramie mosaic virus. We take the method of Proteinase K to extract RNA from the virus extraction and we find a single RNA with the length of 15,000 bp.For the more research on the genome structure of ramie mosaic virus we have established the cDNA library. We use random primer as initiation and AMV Reverse Transcriptase to synthesis the first strand of cDNA, then digest with Rnase H and use DNA polymerase I to synthesis the second strand of cDNA. T4 DNA Polymerase is used to make the ends blunt. And then we add A-tail to the ends using the activity of Taq DNA Polymerase.We use the Resin and Spin Columns of Promega to get off the dATP and small fragment cDNA. After inserting them in pMD-19T vector, we have established the cDNA library of ramie mosaic virus. The average inserts length is 1000kb by the identification of recombinant plasmid PCR and digestion. The blue/white screening we calculate the recombination of the library is 96%. We conclude that the library is qualified for our later research.Select the clones at random and the results of sequencing of B09 and G06 show that: ramie mosaic virus has a more homology with the members of Closterovirus. So we...