[Transgenic tobacco plants with ribosome inactivating protein gene cassin from Cassia occidentalis and their resistance to tobacco mosaic virus].

Abstract

Cassin, the new gene of ribosome-inactivating protein (RIP) isolated from Cassia occidentalis, was inserted into expression vector pBI121 to produce plant expression vector pBI121-cassin (Figs.1, 2). pBI121-cassin was introduced into tobacco cultivar 'K326' by the Agrobacteriurm tumefaciens transformation method and more than 100 independent transformants were obtained. Southern blot hybridization analysis showed that a single gene locus was inserted into the chromosome of the transgenic tobacco lines (Fig.5) and PCR analysis of segregation population of progeny indicated that the inheritance of transgene was dominant in transgenic lines (Fig.4, Table 1). Results of RT-PCR and Northern blot hybridization analysis showed that transgene could be transcribed correctly (Figs.5, 6) . Three self-pollination lines of transgenic T(1) and T(2) were challenged with TMV at different concentration titers by mechanical inoculation. The transgenic lines exhibited different levels of resistance to TMV with the nontransgenic plants. After both titers of TMV concentration were inoculated, transgenic lines were considered as the highly resistant type with a delay of 4-13 d in development of symptoms and 10%-25% of test plants were infected, while nontransgenic control plants were susceptible typical symptoms on the newly emerged leaves (Table 2). One T(2) line, T(2)-8-2-1, was regarded as an immune type because it did not show any symptoms during 70 d and all plants were shown to be virus free by ELISA tests.