Genetic Transformation Of RGSV Genes In Rice

Rice grassy stunt disease is caused by Rice grassy stunt virus (RGSV),it is one of the important rice diseases in tropical and subtropical regions.The symptoms caused by the virus contains increased tillering of rice plants, dwarf, leaf chlorosis and rust. The virus can encode 12 proteins, among which one is RDRP, the other 11 are function proteins. In order to study the relationship between the 11 virus genes and the symptoms caused by the virus in vivo, the individual gene of the virus was transformed into rice.The 10 genes of RGSV were constructed into the carrier PCAMBIA2300-Actin-ocs (PXQ-act), obtaining recombinants PXQ-P1, PXQ-P2, PXQ-P3, PXQ-P4, PXQ-P5, PXQ-P6, PXQ-PC3, PXQ-PC4, PXQ-PC5, PXQ-PC6, and then we transformed them into agrobacterium tumefaciens EHA105 to infect rice callus, followed by screening and differentiation. Finally, the transgenic plants were identified by PCR and Southern blot.As a result, we obtained positive transgenic plants of P2, P3, P4, P5, PC3, PC5, PC6. Tillers of the P3 transgenic plants are significantly increased whoes phenotype is similar to the symptom of rice infected with RGSV, suggesting that the P3 gene may be directly related to the pathogenicity of the virus. Only one PC5 (entatively named PC5-1) transgenic plants showed slow growth, half leaves and leaf curl. The phenotype of other transgenic plants is in accordance with the mock.Real-Time PCR technology was used to detect the mRNA expression level of POL4 and P3 in P3 transgenic plants. POL4 is a kind of gene that closely related to rice tillers. The results show that the gene expression trends of POL4 and P3 in different P3 plants is consistent, while Tiller number are negatively correlated with both of them. At the same time, in order to study whether the symptom f PC5-1 is associated with the expression of PC5 gene, PC5 expression level of normal PC5 transgenic plants and PC5-1 were detected, results show that the PC5 expression levels are of no difference. We then apply the inverse PCR to obtain the insert position of PC5 gene in rice genome of PC5-1, a rice gene of unknown function was found, named nCOX4. After that, we detected the expression level of nCOX4 in PC5-1 transgenic plants, other PC5 transgenic plants and non-transgenic plants, surprisingly to find that the nCOX4 expression level of PC5-1 was significantly inhibited, results show that nCOX4 may be another important gene in rice morphogenesis.