Study On Introducing FMDV Epitopes Fused Gene Into Rice

Plant-derived vaccine is becoming one focus of plant gene engineering, which has such advantages as easy production, low cost, convenient and safe use and easy storage. In this experiment, fused gene O21- O14 -A21-HBcAg containing both type O and A FMDV antigenic determinants were introduced into rice (Oryza Sativa Japonica cultivar Npponbare ) mediated by Agrobacterium.Up till now an effective transformation system has been made, and 193 transgenic seedlings were obtained. PCR, Southern dot blotting, PCR-Southern blotting and GUS gene expression detection has demonstrated their reliability. RT-PCR show the fused gene has been transcribed in the rice. All the work was summarized below:(1) Established the genetic transformation system of Npponbare: The highest transformation frequency was obtained when the transformation process carried at callus pre-dried, Agrobaterium solution concentration reach 0.6 at OD600, the infected time longer than 20min, AS concentration reach 50μmol/L of co-culture medium.(2) Obtain of transgenic seedlings: Altergether there were 207 regeneration seedlings, via PCR, dot blotting, 193 seedlings were considered to be transgenic seedlings. Then the GUS gene expression was confirmation by histochemistry dying. All these reveal that the fused gene had inserted into the genome of Npponbare.(3) Detetion on transcript level: RT-PCR show the fused gene has been transcribed in rice .(4) Obtain of transgenic sub-generation seedlings: Altergether there were 892 seeds of 52 transgenic seedlings.Partial seeds bourgeoned and transgenic sub-generation seedlings were obtained.