STUDIES ON THE RESISTANCE DERIVED BY PVY-C 6kD-NIa GENE

In order to get the antiserum of PVY-C Ma, bacterial expression vectors containing PVY-C 6kD-Nla and VPg gene were constructed, respectively. 6kD-Nla and VPg gene fragments of PVY-C were isolated from pK7 by enzyme digestion and PCR amplification, respectively, then separately cloned into pET-28a. After induced by IPTG, two fusion proteins with size of 27kDa and 25kDa were expressed respectively. The express- -ion product of VPg was purified by SDS-PAGE and electrodialysis, and used to raise against rabbit. The titer of VPg antiserum was determined to be 1:3000 by Western blot analysis. This antiserm also specifically immunoreacted with NIa protein from infected plants by PVY at the position of 49kDa, indicating that the polyclonal antiserum of PVY-C VPg was successfully prepared.The challenge test of 1? and T3 transgenic tobacco plant epressing PVY-C 6kD-NIa gene with PVY-C showed that three types of phenotypes were observed in different transgenic lines. One was that all progenies of each transgenic plant displayed sensitive , which indicating the resistance was lost. The phenotype of resistance, recovery and sensitive occurred together among the progenies in the second type, indicating that the phenotype seperation exist in these transgenic lines. The third type was that all progenies of each transgenic plant displayed resistance and recovery to PVY-C. These results proved that the resistance mediated by 6kD-Nla gene of P\扽-C were stably inherited into T3 generation of the transgenic tobacco plants.PCR and Southern blot analysis indicated that 6kD-Nla gene of PVY-C was not only integrated into tobacco chromosomes but also kept in T3 generration. Western blot analysis showed that 6kD-Nla gene of PVY-C was expressed in three types of transgenic plants. Furthermore, the quantitive analysis revealed that the expression product of PVY-C 6kD-Nla gene was 0.l44%~. 0.086- 0.096%~ 0.110-0.134% of total soluable protein in transgenic plants displaying resistance, sensitive and recovery respectively. Among them, the protein contents of transgene in the resistant transgenic plants were higher than those of the recovered transgenic plants, and the later higher than that of the sensitive transgenic plants. At 38 dpi,the expression product of PVY-C 6kD-Nla gene was highly decreased in both resistant and recovered transgenic plants, even not detected in T3 generation. It remains to further study the real reason to decrease the protein expression of transgene after the inoculation of PVY-C.