Cloning Of Full Length CDNA And Construction Of Infectious CDNA Vector Of Sclerotinia Sclerotiorum Debilitation-associated RNA Virus

Mycovirus are ubiquitous in all kinds of fungi, but it is difficult to discover since mycovirus seldom leads to visible phenotype. There are a few mycovirus in some plant pathogenic fungi that can make their hosts appear hypovirulence. These hypovirus can be used to biocontrol some plant disease successfully, while the vegetative incompatibility obstruct the spread of hypovirus among different strains of pathogenic fungus, then limit the controlling efficiency and application range of these hypovirus. Sclerotinia sclerotiorum, a significant plant pathogenic fungus leads to sclerotium disease on oilseed rape, sunflower and many other important crops around the world. It is difficult to control the sclerotium disease for the short of resistance material, its wide host range, drug resistance and so on. In order to biocontrol sclerotium disease with mycovirus, the Sclerotinia sclerotiorum debilitation-associated RNA virus (SsDRV) of Sclerotinia sclerotiorum hypovirulent isolate Ep-1PN (isolated from eggplant in Jiamusi Heilongjiang province) was cloned and the infectious vector was constructed.The complete nucleotide sequence of the SsDRV of 5419nt, excluding the poly(A) tail, was determined. The sequence was submitted to the GenBank with the accession number AY147260.Sequence analysis revealed the occurrence of a single open reading frame(nt 93-5195) encoded putative replicase protein contained the conserved methyl transferase, helicase and RNA-dependent RNA polymerase (RdRp) domain characteristic of the replicases of potex-like plant viruses (flexiviruses) and Botrytis virus F (BVF), a flexuous rod mycovirus infecting the phytopathogenic fungus Botrytis cinerea. Although phylogenetic analysis of the conserved RdRp motifs verified that SsDRV is closely related to BVF and to the allexiviruses in in the family Flexiviridea, SsDRV is distinct from these viruses, mainly based on the lack of coat protein and movement protein.To clone the fell lengh cDNA, two specific primers were designed beside the single enzyme ClaI (2338nt) site according to the complete nucleotide sequence of SsDRV. One was used to amplify and clone the 3' end region and the other was used to amplify the 5' end region. Two subclonings were ligated at the ClaI site and then the full lengh cDNA were obtained. A trpC promoter derived from Aspergillus nidulans was loaded upstream of the cDNA of SsDRV, and then the casseete was ligated into pCAMBIA1301 vector. At the same time, the CaMV35S promoter which was used to promote hygromycin-resistance gene in the pCAMBIA1301 was replaced with another trpC promoter. In conclusion, the infectious cDNA of SsDRV used for transformation of...