DsRNA-mediated Molecular Immunology Research On Tomato Spotted Wilt Virus (TSWV)

Tomato spotted wilt virus (TSWV) is the type member of the genus tospovirus, the only genus in the family Bunuyaviridae with plant-infecting members. TSWV spreads wide in the world and and caused great economic losses on many crops and plants. what's more, it's difficult to control. Therefore, the author studied TSWV on dsRNA-mediated molecular immunology research to prevent this virus.According to the reported TSWV gene sequence, designed universal primers, amplified the complete sequences of TSWV N gene and GN / GC gene by RT-PCR, cloned into pGEM-T vector. After PCR analysis and restriction enzyme digestion and gel analysis, the results is consistent with expected results. The author got the complete sequences of TSWV N gene and GN/GC gene by DNA sequencing.Compared with other isolates in the GeneBank, TSWV N gene in this study has higher similarity with three isolates from South Korea: up to 99%. And TSWV GN / GC gene has higher similarity with two isolates from Spain: also up to 99%. This result indicated that the materials in this study had occuring genome rearrangement when they infecting the host plants.According to the cloned TSWV N gene sequences, introducting restriction enzyme sites (AscI, SwaI, BamHI, SpeI) to design new primers to amplify 313bp and 536bp fragments in order to construct inverted-repeat dsRNA expression vectors.When the author amplified the N gene reverse fragment, introducted Spel on the 5'end and BamHI on the 3'end, When amplified the N gene forward fragment, introducted Ascl on the 5'end and Sawl on the 3'end. The inverted-repeat dsRNA vector---pFGC1008 was using hundreds of its own sequence as the intron, and the inverted-repeat dsRNA expression vector was using pCAMBIA1304 as plant expression vector. The recombinant inverted-repeat dsRNA vector plasmid is consistent with expected results by PCR analysis,restriction enzyme digestion and DNA sequencmg.The author transformated inverted-repeat dsRNA plant expression vector by agrobacterium-mediated transformation of tobacco, the transforming results need to be further analyzed and identified.