Molecular Diagnosis Of Potato Spindle Tuber Viroid(PSTVd) And CDNA Cloning And Sequence Analysis Of The Isolate From China

All the samples with typical potato spindle tuber viroid (PSTVd) symptoms collected from the fields in the northeast region of China were detected by the R-PAGE method. The positive samples were verified. Then, two-dimensional PAGE and RT-PCR techniques were adapted in deleting PSTVd. The results showed that both 2-D-PAGE and RT-PCR were feasible in PSTVd detection.PGEM-7Z with the monomeric PSTVd cDNA (359 bp) was used as a template and the dUTP was partically replaced by biotin-11-dUTP. The cDNA probe was prepared by PCR amplification technique. Colorimetric and chemiluminescent detection for biotinylated probe were used in this test. They were capable of detecting 50 pg of purified total RNA by a colorimetric assay and 5 pg by a chemiluminescent assay. The nonradioactive probe produced with PCR amplification were particularly suitable for practical diagnosis, as they are sensitive and can be rapidly prepared in large quantities. The two assay methods were 260 times and 26 times as sensitvity as R-PAGE respectively.To device a primer pairs and amplify the full length PSTVd by RT-PCR, the positive RNA extraction from tuber sample was used as template. The product of RT-PCR was purified and connected to the plasmid pMD 18-T vector. Then, the plasmid was transformed into JM109. The full length PSTVd cDNA recombinant plasmid was further identified by restriction mapping analysis and the nucleotide sequence of cDNA cloned in PMD 18-T vector was analyzed with AB1 377 DNA Sequence. Test showed that the cDNA of this Chinese PSTVd isolate was the same as PSTVd-KF-6 mild "Type" isolate which originated from naturally infected potatoes in field of Cornell University Potato Breeding Program. Compared with the mild strain from India and Wisconsin U.S.A., there were only a few mutations in central conserved dormain. That is to say that the PSTVd Strain from China could be a mild strain and there is a high homology in nucleotide sequence with the other mild strains reported. But it was very different mainly in pathogenicity, central conserved and variable dormain from the intermediate and severe strain, for example, PSTVd-F-SL(Severe), PSTVd S27-1-8(Severe) and PSTVd-I-India (Intermediate). It can be futher verified by the test result that the PSTVd found in the northeast region of China may be introduced into China by following the introduction of the potato cultivar Irish Cobbler to various countries and Irish cobbler was imported in Heilongjiang province prior to the 1960s.