P6Protein Encoded By Strawberry Vein Banding Virus Is The Suppressor Of RNA Silencing

Gene silencing is a defense mechanism to counteract the viruses, which is attached to the degradation of exogenous RNA via specific sequences interact ions.Many viruses have evolved suppressor proteins which help them successfully infect their hosts to counteract it.SVBV is a masked virus which can infect strawberry and bring enormous loss to production. P6protein encoded by SVBV P6gene is not only a the symptom determinant, but also the RNA silencing suppressor.In this study, SVBV P6gene and mutant P6gene of Chinese isolate is cloned to the plant expression vector, verifing the suppressed function. In the final, we can get the points of the mechanism of action of P6protein, this will be of great significance to control virus disease by further studying the mechanism on defense and anti-defense between host plant and virus, moreover, our work will be benefit on controlling virus disease as well.1. Construction strategy of plant expression vector containing P6gene and its mutant of SVBV Chinese isolateThe total DNA was extracted from strawberry leaves infected with SVBV Chinese isolate, Specific primers were designed to amplify SVBV P6full length gene and its deletion mutant gene,P6gene and its deletion mutation gene were cloned into plant expression vector pCAM-2300and pGR-106, and positive clones pCAM-CH P6, pCAM-CH△P6, pGR-CH P6, pGR-CH△P6were obtained.2. GFP green fluorescence observation of local silencing experiment and analysis of Real-time PCRPlant expression vector pCAM-CH P6, pCAM-CH△P6were transformed into Agrobacterium tumefaciens EHA105, and co-infiltration was carried out in the leaves of16c N. benthamiana respectively. Agrobacterium tumefaciens containg pCAM-P19and pCAM-2300were controllings. GFP green fluorescence could be stimulated at infiltrating site on leaf and could be observed under UV lamp at6days post inoculation. The results indicated that GFP green fluorescence turned up in the location of the plants infiltrated with pCAM-P19and pCAM-CH P6, but GFP green fluorescence was not observed in the location of the plants infiltrated with pCAM-CH△P6and pCAM-2300.The total RNA was extracted from the location of inoculated paints. Expression of GFP protein was detected by Real-time PCR. The result showed that GFP protein could be detected in the location of leaves of16c N. benthamiana inoculated with expression vectors pCAM-P19and pCAM-CH P6, but GFP protein could not be detected in the location of leaves of16c N. benthamiana inoculated with expression vectors pCAM-CH△P6and pCAM-2300.3. The rate of systemic silencing, reversing exeriment and observation of GFP green fluorescencePlant expression vector pCAM-GFP was transformed into Agrobacterium tumefaciens, and co-infiltration was carried out in the leaves of2316c N. benthamiana respectively. Observed under UV lamp at15days, systemic silencing occurred in1516c N. benthamiana. GFP green fluorescence could not be observed under UV lamp at30days post inoculation, and the rate of systemic silencing is60.87%. The rest916c N. benthamiana had no changement.The expression vector pGR-CH P6and pGR-CH△P6were transformed into A. tumefaciens EHA105, respectively, and co-infiltration was carried out in the leaves of16c N. benthamiana plants. Mild mosaic symptom could be observed at15days post inoculation. GFP green fluorescence could not be observed under UV lamp.4. GFP green fluorescence observation of systemic silencing experiment and analysis of Real-time PCRPlant expression vector pCAM-CH P6, pCAM-GFP were transformed into Agrobacterium tumefaciens EHA105, and co-infiltration was carried out in the different leaves and different location of the same leaves of16c N. benthamiana respectively. GFP green fluorescence of the aptical leaves could be observed under UV lamp at15days post inoculation, and the results indicated that silencing turned up in the plants, which were infiltrated with pCAM-GFP at upper leave, s with pCAM-CH P6and pCAM-GFP infiltrated at bottom of leaf, pCAM-CH P6infiltrated at tip of leaf.The total RNA was extracted from the aptical leaves of inoculated paints. Expression of GFP protein was detected by Real-time PCR. The results indicated that GFP protein could be detected in the plants, which were infiltrated with pCAM-CH P6at upper leave, with pCAM-GFP and infiltrated with pCAM-CH P6at bottom of leaf, pCAM-GFP infiltrated at tip of leaf. But GFP protein could not be detected in the plants, which were infiltrated with pCAM-GFP at upper leave, s with pCAM-CH P6and pCAM-GFP infiltrated at bottom of leaf, pCAM-CH P6infiltrated at tip of leaf.