Transformation Of Nicotiana Tobacum And N.benthamiana With Potato Virus Y HC-Pro And Its Mutants

The helper component proteinase (HC-Pro) is a key protein encoded byplant viruses of the genus Potyvirus. HC-Pro can be schematically divided intothree regions: an N-terminal region essential for the transmission process, aC-terminal region harboring the proteinase activity, and a central regionimplicated in other functions. The transmission function involves twoconserved motifs. One is the N-terminal KITC motif that is involved inbinding to the aphid vector's stylets. The other is the C-terminal PTK motif,which contributes to binding of HC-Pro to the viral capsid protein'sN-terminal DAG motif. In our study, a mutant which PTK motif was deletedwas constructed and transformed to tobacco (NC89). We proved the deletionmutant could compete with wild HC-Pro and reduce the efficiency oftransmission.RNA silencing is due to sequence specific RNA degradation in thecytoplasm in eukaryotes. Transgenic plants expressing virus genes or sequenceshave been shown to be resistant to plant virus infection. More and moreresearches show that dsRNA maybe the trigger of RNA silencing. We hereconstructed single strand sense gene structure and inverted repeat genestructure from PVYN HC-Pro.The results and conclusion are as follows:(1) The N-terminus of PVYN HC-Pro (N1) was amplified by PCR and inserted into clone vector pUC18. The cloned translatable N1 was confirmed by sequencing and ligated into downstream of 35S promoter in the binary vector pROKII to generate pROKN1. The recombinant plasmids were introduced into Agrobacterium tumefaciens LBA4404.(2) The translatable N1 was inserted into protokaryote expression vector pET22b(+) to generate pETN1. After induced by IPTG, N1 could express in E.coli BL21.(3) pROKN1 was introduced into tobacco plants (NC89) via Agrobacterium tumefaciens -mediated transformation. After tissue culture, we obtained 253 regeneration plants. The results indicated that 98% of the plants (248) transformed were transgenic plants. After inoculation, we found most of transgenic plants show susceptible phenotype, only 3 of 248 show resistance to PVYN. Aphid transmission assay indicated that the transgenic plants could reduce efficiency of virus transmission by aphids significantly.