Function Of P6Protein In The Pathogenic Process Of Strawberry Vein Banding Virus

The pathogenicity of plant virus is often closely related with the symptom determinant encodedby virus genome, that is to say the symptom determinant can help virus infect host successfully,make the host diseased even dead. P6gene of SVBV Chinese isolate was cloned in our researchwork. Nicotiana benthamiana was inoculated with virus vector carrying P6gene or mutant P6gene, and N．benthamiana and N. tabacum were transformed with binary expression vectorcarrying P6gene. The role of P6in the progress of virus infection and symptom formation andthe function of SVBV P6as a symptom determinant could be determined by observing thesymptom phenotype of inoculated and transgenic tobacco plants. Our research work will finallyascertain pathogenic mechanism of SVBV on molecular level. This will be of great significancein further studying molecular mechanism on defense and anti-defense between host plant andvirus, moreover, our work will be benefit on controlling virus disease as well.1. Cloning and sequence analysis of P6gene of SVBVThe total DNA was extracted from strawberry leaves infected with SVBV, Specific primerswere designed to amplify SVBV P6full length gene, then the gene was cloned and sequenced.Sequence analysis showed that the full-length of P6gene of SVBV Chinese isolate was1557bp,encoding518aa. P6gene of SVBV Chinese isolate was compared with P6gene of SVBVAmerican isolate and P6gene of the other members of Caulimovirus, the result showed that P6gene of SVBV Chinese isolate shared the highest sequence similarity (84.5%) with that of theSVBV American isolate, while had relatively lower nucleotide sequence similarity(23.7%~27.9%) to P6gene of the other members of Caulimovirus. A phylogenetic tree based onalignment of P6gene nucleotide sequences of SVBV and the other members of Caulimoviruswas constructed. The result indicated that SVBV Chinese isolate and SVBV American isolateclustered into a separate branch. And it illustrated that the two SVBV derived from strawberryhad the closest relationship but they had relatively distant relationship with the other members ofCaulimovirus. The data obtained by bioinformatics analysis demonstrates that the P6proteinexpressed by P6gene of SVBV contains many kinds of protein kinase phosphorylation site, andthe secondary structure of P6contains a α-helix-rich region at its N-terminus.2. Construction of plant expression vectors of P6gene and its deletion mutant genes ofSVBVP6gene and its deletion mutation genes of two isolates of SVBV were cloned into virus vectorpGR106, and positive clones pGR-CH P6，pGR-US P6，pGR-CH△P6and pGR-US△P6wereobtained. P6gene of two isolates of SVBV were cloned into binary expression vector pCAM2300, and positive clones pCAM-CH P6，pCAM-US P6were obtained. 3. Transient expression was conducted by transforming expression vectors viaAgrobacteriumExpression vector pGR-CH P6，pGR-CH△P6，pGR-US P6，pGR-US△P6and empty vectorpGR106were transformed into Agrobacterium tumefaciens, and N. benthamiana leaves wereinoculated with A. tumefaciens harboring these expression vectors, respectively. Symptom ofsystemic leaves of N. benthamiana was observed15days after inoculation. The systemic leavesof N. benthamiana inoculated with expression vectors pGR-CH P6，pGR-US P6showed seriousmosaic, aptical leaves crincle, leaf curl symptoms. The systemic leaves of N. benthamianainoculated with expression vectors of pGR-US△P6，pGR-CH△P6showed mild mosaicsymptom, and aptical leaves can unfold basically and do not show obvious leaf curl symptom.The systemic leaves inoculated with empty vector pGR106just showed the typical mild mosaicsymptom of virus PVX, and aptical leaves develop normally.4. Molecular detection of P6gene transient expression in N. benthamianaThe total RNA was extracted from inoculated N. benthamiana, and specific fragment ofvirus PVX and P6gene were detected by RT-PCR. The result showed that specific fragment ofvirus PVX and P6gene fragment could be detected in systemic leaves of N. benthamianainoculated with expression vectors pGR-CH P6，pGR-CH△P6，pGR-US P6，pGR-US△P6.However, only specific fragment of virus PVX could be detected in systemic leaves of N.benthamiana inoculated with empty vector pGR106but fragment of P6gene could not bedetected. Furthermore, semi-quantitative RT-PCR was carried on detecting P6gene of N.benthamiana. It was found that RNA transcription level of P6gene have not any obviousdifference in N. benthamiana inoculated with the expression vectors carrying P6gene and mutantP6gene.5. The impact of transient expression of P6protein on PVX duplication in N. benthamianaN．benthamiana were inoculated with pGR-CH P6、pGR-CH△P6and pGR106, respectively,the duplication quantity of PVX in systmic leaves of N．benthamiana was detected by sandwichELISA. The result showed that N．benthamiana inoculated with pGR-CH P6has the highest PVXduplication quantity which increased60.7%than that of N．benthamiana inoculated with emptyvector pGR106. The duplication quantity of PVX have no significant difference inN．benthamiana inoculated with pGR-CH△P6and control pGR106.6. Molecular identification and symptom observation of transgenic tobacco plantsExpression vector pCAM-CH P6was transformed into A. tumefaciens, and N. benthamiana andN. tabacum were transformed via leaf discs. The total DNA was extracted from transgenictobacco. The transgenic tobacco plants were detected by PCR using specific primers. It wasproved that P6gene of SVBV Chinese isolate had already been transfered into N. benthamiana and N. tabacum. It was found that the transgenic N. benthamiana showed up-curl symptom ofaptical leaves. Otherwise, transgenic N. tabacum display no obvious disease symptom comparedwith wild type N. tabacum.7. Detection of P6protein in transient expression and transgenic tobacco plantsIt was found that P6protein could accumulate abundantly in the systmic leaves of N.benthamiana inoculated with pGR-CH P6by Western blotting. Whereas, the accumulationquantity of P6protein in N. benthamiana inoculated with pGR-CH△P6decreased dromatically,and P6protein coult not be detected in N. benthamiana inoculated with empty vector pGR106.P6protein could be detected in transgenic N. benthamiana plants, but its accumulation quantitywas very low. The accumulation of P6protein of transgenic N. benthamiana plants was evenlower than that of N. benthamiana inoculated with pGR-CH△P6, and the accumulation of P6protein of non-transgenic N．benthamiana plants could not be detected.