Cloning The 5'-End Sequence And Construction Of Full-Length CDNA Infectious Vector Of Sclerotinia Sclerotiorum Virus SsDRV

In this study, the full-length genomic sequence of Sclerotinia sclerotiorum debilitation associated RNA virus (SsDRV) modified. A cDNA infectious vector of SsDRV was constructed, and the infectious cDNA vector was transformed into Sclerotinia sclerotiorum strain Ep-1PNA367 (virus free).Previously, a putative full-length cDNA of SsDRV was cloned onto pCAMBIA1301 vector, named as p1301-SsDRV (Xie et.al, 2006). In this study, pl301-SsDRV was used to transform Coniothyrium minitans, a mycoparasite of S. sclerotiorum with Agrobacterium tumefaciens-mediated transformation. 105 transformants were obtained; some of them were comfirmed PCR amplification. The colony morphology, conidial production and parasitical ability of some transformants were studied, and the results suggested that the phenotypes of transformants were not significantly affected. The viral cDNA could be detected from tested transformants, but dsRNA could not be extracted from them with cellulose CF-11. Thus, the full-length cDNA clone of SsDRV was in doubt. The full-length cDNA of SsDRV was re-analyzed, and the results suggested that the former spliced cDNA was incomplete, and the sequence of 5' end there might be missed..An additional 82 nt-length fragment at the 5' terminus was cloned from total RNA of strain Ep-1PN with SMART (Switching Mechanism At 5' end of the RNA Transcript, SMART) technique. Thus, the genome of SsDRV was re-spliced, and the full-length sequence is 5500 nucleotide (excluding polyA) with only one open reading frame (174nt-5274nt), there is a cap structure at 5' end. Further analysis showed that the sequence is varable at the 5' untranslation region, and has a polyA structure at 3' end. The secondary structures of 5'-UTR and 3'-UTR were predicted, and three hairpin structures were deduced at 5'-UTR and a tRNA knot structure at 3'-UTR.Based on the modified genomic sequence of SsDRV, a new full-length infectious cDNA vector, named pTSVH, was constructed by inserting the cDNA between trpC promoter and trpC terminator from Aspergillus nidulans. The pTSVH was linearzed with restriction enzyme, and transformed into S. sclerotiorum strain Ep-1PNA367 with electroporation using protoplast as acceptor, 9 transformants were obtained. The growth rate and the pathogenicity to tomato leaves of the transformants were reduced slightly. PCR analysis showed that S.sclerotiorum Ep-1PNA367 had the cDNA of SsDRV. RT-PCR analysis showed that the cDNA of SsDRV had transcribed normally, but the replicating form (dsRNA) could not be detected from these transformants.