Coat Protein Gene Sequence Analysis And Strain Identification Of A Potato Virus X Isolate

Transgenic plants expressing virus genes or sequences have been shown to be resistant to plant virus infections. This form of resistance is known as pathogen-derived resistance (PDR). Viral coat protein gene has become a standard strategy in anti-virus breeding. The analysis of resistance of plants transformed viral cp gene showed that RNAs mediated plant virus resistance. Due to its advantage of high bio-safety and effect, the RNA-mediated resistance now becomes a hot spot in the domain of plant anti-virus gene engineering. In this research, an isolate of PVX from Shandong was obtained and its CP gene was cloned by using RT-PCR method. The strain identification was due to the analysis of nucleotide sequence and deduced amino acid; meanwhile, we conducted a survey of potato plants carrying PVX in fields in Shandong; we also transformed the NC89 with untranslatable coat protein gene to generate RNA-mediated resistant transgenic tobacco plants. The main results presented in this thesis are as follows:1 An isolate of Potato Virus X was obteined from potato cultivated in Shandong Province which was named PVX-SD1. The shape of virus particles is filament-like. The subunits' molecular weight of the coat protein was 28.2kD .The coat protein gene of the PVX was amplified from the extracted viral RNA by using reverse transcription-Polymerase chain reaction(RT-PCR) method, and cloned into plasmid pUC19.The nucleotide sequence of the CP gene was determined and sequence analysis showed that the CP gene of PVX-SD1 included 719bp, encoding 238 amino acid. The sequnce of PVX-SD1CP gene was submitted to the NCBI,and obtained an registration number: AF528555.2 The nucleotide sequence of the PVX-SD1 CP gene was compared with other isolates reported in GenBank. It shares 80.1%～99.7% and 89.8%～100% homology at the sequence levels of nucleotide and deduced amino acid, respectively. PVX-SD1 shares 99.7% and 100% homologies at the nucleotide and deduced amino acid levels, respectively, with one of the European strain UK3, indicating that they have the same response on potato.3 PVX-SD1 was purified with high yield using an improved purification procedure. We carried out proper injection process and got high specificity antiserum to PVX, the titer of the antiserum was 1/2048 . The antiserum can be used to diagnose and analyze plants infected with PVX.4 Using random sampling and ELISA, we conducted a survey of plant carrying PVX in fields. The result shows that 15%--29% of potatos were infected by PVX,and always coinfected with other plant viruses.5 The untranslatable CP gene was cloned and inserted between the 35S promoter and nos terminator sequences of the binary vector pROKⅡ,The recombinant plasmids were introduced into Agrobacterium tumefaciens LBA4404 using freezing & melting method. The transgenic tobacco plants were obtained via Agrobacterium -mediated transformation. Based on the virus infection assay, we identified the resistant plants. The PCR assay showed that the transgene had integrated into the tobacco geonme.