| Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yipinhong with yellowing at leaf edge in China. Purified viral particles of 28-30 run in diameter contained dsRNA segments with the same number and mobility as those extracted directly from radish leaves, indicating all the four dsRNA segments were of viral origin.A modified single primer amplification technique (SPAT) and RT-PCR were adopted to get the cDNA of all dsRNA segments, followed by cloning and sequencing. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained an ORF on their plus-stranded RNA, putatively encoding a RdRp (66.9 kDa) and a CP (55.9 kDa) similar to that encoded by members of the family Partitiviridae. Particularly, 6 conserved motifs specifically harbored in RdRp of members of Partitiviridae, were also showed in RasR 1 -encoded RdRp. In addition, both RasR 1 and RasR 2 were highly conserved at the 5'UTR and had an adenosine-uracil (A/U) rich stretch at the 3' UTR respectively, with an identical terminal motif (5'-AAAAUAAAACC-3'). According to the electrophoretic profile of the 5% polyacrylamide gel, the content of thetwo minor dsRNA bands were much weaker than the two major. cDNA cloning and sequencing revealed that the smaller of the two minor bands is actually a doublet consisting of two co-migrating dsRNA segments and the resulting three segments were designated as RasR 3, RasR 4 and RasR 5. RasR 3 was 1717 bp in length and potentially encoded a protein of about 55.3 kDa, containing all of the six conserved motifs shared by the RdRps of members of the family Partitiviridae. RasR 4 and RasR 5 were 1521 and 1485 bp in length and each encoded a putative protein of unknown function. Their molecular masses, as calculated from the deduced amino acid, were 38.2 kDa and 38.8 kDa, respectively. Comparison results showed that RasR 4- and RasR 5-encoded shared some similar regionsin their sequences. The 5' UTRs of all three segments shared regions of high sequence similarities, with an identical terminus (5'-GAUAAUG-3'), which was distinct from those of the RasR 1 and RasR 2. Plus, none of these three segments has an A/U rich tail at the 3' UTR and their 3'UTR sequences were distinct from those of RasR 1 and RasR 2, too. Taken together, these results suggest that more than one partitivirus were co-infecting radish leaves. Considering the fact that RasR 1 and RasR 2 could independently exist in the host, the results of sequence comparison and the identification of the family Partitiviridae by ICTV, the two major dsRNA segments might constitute the genome of an unknown member of this family, tentatively named as Raphanus sativus virus 1 (RasV 1). RasR 3 is suggested to come from anther unknown member with the temporary name Raphanus sativus virus 2 (RasV 2). Considering the similarity RasR 4 and RasR 5 share with RasR 3 at their UTR, and the situation that RasV 2 still need a dsRNA segment encoding its CP, we suggest that RasR 4 or RasR 5 is RasV 2's CP-encoding segment. On the other hand, the possibility that they are satellite RNA supported by the viruses should not be excluded.
|
PDF Full Text Request