Detection And Identification Of The Viruses Infecting Panax Notoginseng

It was shown that there were a potyvirus and at least two other viruses in the diseased leaves of Panax notoginseng showing crimple symptom by biology assay, serology, electron microscopy observation and molecular biology method. ToMV had ever been detected by EL1SA and RT-PCR analysis, but it was not the main pathogen.Seventeen indicator plants belonging to 4 genuses were mechanically inoculated with the leaf sap of diseased P. notoginseng, and all showed no symptom. When indicator plants were inoculated with the leaf sap of diseased P. notoginseng and Nicotiana tobacum leaf infected by TMV or the leaf sap of diseased P. notoginseng and Cucurbita pepo leaf infected by ZYMV, the severity of the symptom was in close relation with the concentration of the leaf sap of P. notoginseng. So it was testified that there is some substance in P. notoginseng that can inhibit the infection of virus.The viruses in P. notoginseng were not serologically related to all the 29 antisera of 14 genuses by ELJSA analysis.The dsRNA was extracted from diseased leaves of P. notoginseng, and a cDNA library was obtained after reverse transcription. Twelve clones were randomly selected to sequencing, and sequence analysis revealed that the sequences of 3 clones had relatively high homology with potyviruses. The sequences of other 9 clones had homology with Rice ragged stunt virus (RRSV), Saccharomyces cerevisiae virus L-A and Oyster mushroom isometric virus respectively.No Phytoplasma and viroid was detected by nested-PCR and bi-directional SDS-PAGE.The antiserum of ZYMV was prepared and provides condition for the identification of ZYMV. The CP gene of ZYMV was amplified by RT-PCR, and ligated to the expression vector of pET-22b(+) . The recombinant plasmid pET-ZCP was transformed into E. coli BL21 (DE3) and then induced by IPTG to express a fusion protein .It was showed by SDS-PAGE and Western blot analysis that the CP gene was expressed at high level .The molecular weight of the fusion protein was about 33.0kD . An antiserum with high specificity was produced after the rabbit was immunized with purified fusion protein, and its titer was determined to be 1/4096 by ACP-ELISA.