Transformation Of SP Gene Of Rice Grassy Stunt Virus And Disease Resistance Analysis

Large amount of disease-specific protein (SP) accumulated in the rice plant cells infected by Rice grassy stunt virus (RGSV). The protein was encoded by SP gene on vRNA6 and presumed possibly pathogenicity related to protein. But its function is not clear. In this study, RGSV SP was transformed into rice by Agrobacterium-mediated rice transformation. Analysis of different phenotypes and disease resistance has been performed in order to further study the function of SP gene and its interacation with host gene.With primers desinged according to sequence of RGSV-IR isolate, the cDNA clone of SP of RGSV-SX isolate was obtained by RT-PCR. Sequence analysis showed SP gene is composed of 540bp, including A 33.5%, C 17.7%, G 20.7%, T 28.1%. The nucleotide sequence homology of SP gene was 100%, compared with the IR isolate that has been published.The SP gene was identified by XbaI/SacI or XhoI/SacI or BamHI/SacI, and then linked to the plant expression vector pEmu-mcs-N or pKYLX71:35S2 or pCBTNSv6 respectably which was also digested by Xba I/SacI or Xhol/Sacl or BamHI/SacI. The pEmu-SP, pK-SP and pCB-SP were constructed and introduced into Agrobacterium by DNA-directed transformation or triprental mating separately.The above plant expression vector were used in Agrobacterium-mediated rice transformation system for cultivars of Japonica rice, including Tainong67, Jinnan8. After the co-cultivation, selection and regeneration, about 58 regenerative rice plants were obtained. PCR PCR-Southern hybridization showed SP gene has been transferred into 31 transormed rice plants. PCR-SSCP and sequencing assay showed that the target gene is complete in transgenic rice plants. Northern blot hybridization showed the target gene has been transcripted into mRNA. ELISA or Western blot showed that SP gene has been expressed in 3 transgenic rice plants. It showed primarily that the symptom of rice can not be caused by SP separately. Disease resistance analysis showed that some transgenicrice plants got certain resistance to RSV, reduction in virus accumulation and delay in symptom development. And the disease resistance was deduced to mediate by RNA.Genetic analysis and PCR-Southern of T1 trangenic rice plants showed that the target gene can pass from TO to T1, and genetic segregation accorded with 3:1 and 15:1 Mendelian segregation ratios. Further study on disease resistance to RSV showed that segregation and reduction of disease resistance happened in different lines and plants of Tl transgenic rice plants.