Antiserum Preparation And Identification Of RNA Silencing Suppressor Of LSV 16KDa/TGB1

Lily symptomless virus (LSV) is belong to carlavirus, which is one of the three viruses infecting lily and it can influence the quality of bulb and freshly cut flower badly.The genome of lily symptomless virus is single-stranded positive RNA molecule which contains six open reading frames and it can encode four proteins from 5r to 3':RdRp, TGB1-3, CP and 16kDa.The leaves of lily which were infected LSV were used to clone LSV 16kDa by RT-PCR in the study. The fragment was ligated with expression vector pET-28a(+), then transformed into E.coli BL21(DE3). Protein was overexpressed by IPTG induction. The high expression of 16kDa protein which existed in a form of inclusion body detected by SDS-PAGE and analysised by mass spectrographic. The recombinant protein was purifed through Ni-chelating affinity chromatography and obtained high purity. Antibodies were separately prepared with the mices which were immunized by the purified recombinant proteins acted as antigens. Specific antisera against the fusion protein were detected by indirect ELISA and western blot analysis using specific antisera showed the specific reaction to the proteins of 16kDa, TGB1 and leaves of lily because of appearance of specific protein band. These results demonstrated that antisera can be used for the detection of virus, western blot and immunohistochemistry.In order to prove which protein encoded by LSV can suppress RNA silencing, The TGB1, 16kDa and 16kDa? separately were cloned into pMDTM18-T simple vector, meanwhile sequenced exactly. Then they separately were inserted into expression vector PTF101.1, and the recombinant expression vectors were constructed. Agrobacterium tumefaciens strain GV3101 was transformed with recombination plasmid, then infected N. benthamiana 16c. The TGB1 and 16kDa separately were cloned into binary expression vector pGR107 and infected N.benthamiana. The study showed that 16kDa can inhibit GFP-mediated local and systemic gene silencing, reverse systemic RNA silencing and cannot inhibit local GFP silencing triggered by dsRNA. Meanwhile TGB1 cannot be a RNA silencing suppressor. In addition,16kDa, TGB1 cannot enhance the pathogenicity of PVX and synergistic effect.In addition, PTF101-16kDa/TGB1 were separately infected by agrobacterium mediated methods into common tobacco and lily, meanwhile mitochondria proteins were extracted in tobacco and lily. It turned out that 16kDa and TGB1 were located in cell mitochondria by western blot. And 16kDa and TGB1 were located in chloroplast by immunogold electron microscopic localization, but content of gold particles was low. These results demonstrated that 16kDa and TGB1 were mainly located in mitochondria and had limited effects on chloroplast.In conlusion, this study demonstrated that 16kDa was a RNA silencing suppressor by functional analysis of LSV 16kDa and TGB1, meanwhile 16kDa and TGB1 were mainly located in mitochondria. The outward symptom of lily infected by LSV is not obvious, protein receptors of 16kDa and TGB1 may be not chloroplast but mitochondria.