Molecular Variability Of The Coat Protein Gene Of Potato Virus Y

Potato virus Y (PVY) is a very important plant virus that can infect tobacco and cause serious econimic loses. The research of molecular variability of PVY have long history, but only a few PVY CP gene from CHINA have been sequenced, so the main objective of our experiments was to iditify the biological symptoms, molecular variability, strain type and phylogenetic relationship of different PVY CP genes using the materials of 108 field tobacco isolates which were infected by virus. At the same time, the other objective is the production of PVY antiserum that is the foundation of experiment based on antibody.PVY were purified with the procedure including PEG precipitation and differential centrifugation. The purified PVY that have typical flexuous rods particles were seen by electron microscopy. Virus coat protein migrated as a single component in SDS-PAGE with MW of 20 KD.Rabbit was immunized using the purified virus and the antiserum was obtained after the fifth immunization.Indirect ELISA assay showed the antiserum had titer of 1: 8192 with high specificity to PVY.Western bloting results combined with Indirect ELISA results showed that the PVY antiserum have no serological reaction with RSV, SCMV and health tobacco extract. The highly specificity PVY polyclonal antibody lay foundation for the serological reseach methods.Thirty-three tobacco samples showing tobacco necrosis diseases were obtained from tobacco field representative of ten regions in China. Indirect-ELISA were done using the PVY antiserum to detect the all the 108 PVY field isolates. The results shown 33 isolates have positive reaction with PVY antiserum. The health tobacco were inoculated by those 33 isolates and shown systemic symptoms including two main symptoms: vein necrosis and mosaic. All the 33 isolates sequenced in this study had the capacity to infect tobacco systemically in greenhouse, causing two main symptoms: vein necrosis and mosaicThe result of Indirect ELISA showed they were all infected by Potato virus Y (PVY), and then the nucleotide sequences and predicted amino acid of the coat protein (CP) genes of PVY tobacco isolates from different regions of China were determined. Comparison of all sequenced isolates revealed high conservation of the CP gene. The CP gene nucleotide sequence showed more than 83.2% identity with all the PVY isolates sequenced and the amino acid sequence identity is between 93.7 to 100%. The highest degree of identity was observed in the C-terminal half where only 11 amino acid substitutions could be observed in contrast to the N-terminal half with 22 substitutions.