Rice Transformation Of RIP Gene From Gynostemma Pentaphyllum

Ribosome-inactivating protein (RIP) is a sort of toxin protein existing widely in higher plant which can inhibit the synthesis of protein. Lots of research show that RIP possesses the resistance to bearing, tumor and human virus in medicine. Some RIP had applied in clinic well. On the other hand, RIP hold the resistance to plant virus, pathogen fungi and pest in plant protect. Therefore, the research to RIP has great medicinal and agricultural appliance.Gynostemmin is a type I RIP from Gynostemma pentaphyllum. Its gene was ligated with the plant expression vector pCAMBIA1300-CP. The recombinant vector was named pCA1300-RIP. After tri-parental mating, the RIP gene was introduced into Agrobacterium twnefaciens LBA4404. The immature embryo, subcultured immature embryo and mature embryo of Japonica rice Tai-nong 67 were used as explants for calli induction. After co-cultured with recombinant Agrobacterium tumefaciens, the calli were screened and differentiated under the pressure of Hygromycin B, and transformational plants were got after regeneration. The genome DNA was extracted and used as template for PCR and PCR-Southern dot blot. The results show that the target gene had integrated into the genome of rice. And the PCR-Southern blot analysis suggests that the amplification fragment is the foreign sequence but not the endogenous sequence of rice. The results of RT-PCR and RT-PCR-Southern blot show that the target gene had been transcripted.After Hygromycin B resistance selection, the character separation of Hygromycin resistance takes place in seeds of TO plant. However, the seperation rate is not consistant with the Mendal seperation rate. We speculate that the target gene had lost during the genetics progress in some rice or that the target gene affacted the metabolization of rice, therefore the rice was endowed with the Hygromycin resistance.The fused gene of RIP-GST was induced with IPTG, and the expressed production was used as antigen for immunity to rabbit. The titer of the antiserum is 2-11 detected with indirect ELISA. The antiserum can be used for Western blot.