Expression Of Translatable And Untranslatable Potato Virus Y Coat Protein Genes In Tobacco And Resistance Comparison Between The Transgenic Plants

Transgenic plams expressing virus genes or sequences have been shown to be resistant toplan virus infections. This fOrm of resistance is known as pathogen-derived resistance (PDR).Viral coat protein gene has been widely used fOr PDR, bllt the mechanism of resistance(protein-mediated or RNA-mediated) has not been yet comPletely understood. AsRNA-mediated resistance shows several advantages over protein-mediated resistance,therefOre, RNA-mediated resistance has been widely studied in recent years. We here describethe transformation of tobacco (cultivar NC89) with the untranslatable coat protein gene fromPVY strain N (PVY' CP) to generate RNA-mediated resistam tYansgenic tobacco plams.MeanWhile, we also transformed the NC89 with tYanslatable PVYN CP gene to determinewhether the observed resistance is caused by expression of the viral CP gene on the ANA oron the protein level. The main results presented in this thesis are as fOllows:l. According to pubIished nucleotide sequence, the translatable and ulltranslatable coatprotein (CP) genes of PVYN were synhesized by RT-PCR and inserted into clone vectorpBSK. The untranslatable cDNA was designed in such a way that a base (T) was insertedthree bases after the translation start codon (ATG), fOrming a stop codon (TGA). The clonedcDNAs of translatable CP(CPN) and untranslatable CP(CPM) were inserted between the 35Spromoter and the nos terminator sequences of the binary vector pROKII to generatepPVYCPN and pPVYCPM, respectively. The recombinant plasmids were introduced intoAgrobacterium tumefaciens LBA4404 using tri-parental mating.2. The translatable and the unranslatable gene was introduced into tobacco (NC89)pltals via Agrobacterium tumefaciens-mediated transformation. The tYansformed tissue wasselected in the presence of l50mg/L kanamycin sulfate and regeneration plants initially werescreened by Dot-blot. The results indicated that 95% of the plams ttansfOrmed withtranslatable PVYN CP gene and 98% of the plans transformed with untranslatable PVYN CPgene were Dot-blot positive. All of the progeny To transgenic plans aPpeared normal inmorpho1ogy and development.3. Based on the virus infection assays, we idelltified three kinds of phenotypes to viralinfection: (l) the plants were susceptible, which developed very severe symptoms and werestunted and heavily mottled, in some case the plans were killed; (2) the plams were partiallyresistan, the plams showed light symptoms 20-30 days post inoculation; (3) the plans werehighly resistani, no virus replication occurred. The resistani assays also showed that theproportion of highly resistance plants were about 8%. The resistant plants were also effectiveagainst PVYN ANA, and it is not broken with increased dose of the purified virus. The highlyresistance plans transfOrmed with pPVYCPM were also highly resistam to PVYo infection.4. Genomic DNA from the progelly To transgenic plans and noniTansgenic plans wasdigested by KPnI. The results of southern blot analyses indicated that the foreign gene hadbeen integrated iflto the tobacco chIomosomes. Further experiment indicated that theresistance of the two kinds of progeny To transgenic plants was all related to copy numbers ofthe transgenes. The highly resistant planis contained 5-6 copies of the transgene, the pndia1lyresistam plants contained 3-4 copies, and the susceptible plams colltained 1 -2 copies.5. Northem b1ot analyses of leaf samp1es taken prior to mechanical virus inoculationshowed that a1l progeny To transgenic plaflts expressed the PVYN CP anA, and the levels oftranscript accumulation varied among transgenic lines. The highly resistam planis had alow-level accumulation of the PVYN CP Whereas, the PVYN CP ANA in suscePtible plantswas accumulated to higher 1evels than that in the high1y resistant plants. The results revealedan inverse correlation between tYansgene transcript accumulation and virus resistance.6. Western blots indicated that th...