Transformation Of SP Gene Of Rice Stunt Grassy Virus And Its Transcript And Expression In Rice Protoplast

Rice grassy stunt v/ray(RGSV), a member of Tenuivirus with a lot of unique characteristics in genomic structure and biological property, includes six ssRNA which have ambisense coding strategy. Now the total genome of RGSV had been determined, but the function of proteins is not clear.Disease specific protein (SP) encoded by vRNA6 gene of RGSV is presumed possibly pathogenicity related to protein. It is also reported that SP is accumulated in infected rice but little accumulated in insect. It shows that SP take an important role in the host. But the mechanism is not clear. The transgenic technology was used to investigate the function of SP in the host and definitude the relation between SP and the pathogenicity. In order to further research on the regulation period of SP gene, we detected the transcript and expression of SP gene in infected protoplast.The target gene was introduced into Agrobacterium LBA4404 by triparental mating. The acceptors were tender embryo, mature embryo and froliferous embryo. Then transformation was done by Agrobacterium-mediated system. PCR analysis and southern blot hybridization showed primarily that SP gene has been transferred into rice. And it also showed that the target gene in some regenerative plants has been transcripted into mRNA by using RT-PCR southern blot hybridization. It showed primarily that the symptom of rice can not be caused by SP.In order to further research on the regulation period of SP gene, the transcript of SP had been detected by Northern blot hybridization after RGSV infected protoplast. A rice protoplast system that can be infected by RGSV has been established. Following Poly-L-Lysine mediated, rice protoplasts were inoculated with purified RGSV. Protoplast samples were collected at various time and hybridized to DIG-labbed riboprobe. Northern blot analysis showed that the accumulation of mRNA could be detected after 8h, its peak came to a head in 24-32h, and then began to decrease, but it still had accumulation. The expression of SP was detected by Dot-ELISA, the result showed that SP began to express in 12h and came to a head in 32h, then decreased gradually.