Identification Of Infectious Clone And Functional Analysis Of Transactivator Of Strawberry Vein Banding Virus

Chinese isolate of Strawberry vein banding virus(SVBV)infectious clone pSVBV-SY of has been shown to infect forest strawberry,but the low infection rate,hindering the study of infectious clones.In this study,the efficiency of inoculation was improved by vacuum inoculation of strawberry.pSVBV-SY was highly infect in forest strawberry and showed typical vein banding symptom.The same vaccination method was used to inoculate the SVBV virus vector pSVBV-MCS on forest strawberry to verify its infectivity.The study has found that the P6 protein of virus of Caulimoviridae has been shown to be able to transactivate the translation of polycistronic mRNA.This study also identified the SVBV virus P6 protein is a transactivator,which can efficiently activate the expression of the second gene of bicistronic.To predit the functional domain of P6 to find the core domain of transactivator,construct series P6 mutants.The results showed that all mutants of P6 could not transactivate the expression of the second gene of bicistronic.1.Infectivity identification and detection of SVBV infectious clonesComparison of scratch inoculation and inoculation efficiency of vacuum filtration method with strawberry,SVBV Shenyang isolate infectious clone pSVBV-SY inoculated forest were inoculated with 15 strains of strawberry,after 35 d detected infection rate,the infection rate of inoculation scratch only between 20%-40%,vacuum inoculation infection rate between 86%-100%.The pSVBV-SY IV gene and ORF VI gene were detected in the ORF of the susceptible plants PCR.Southern blot(DNA)was used to detect the DNA of strawberry,which further confirmed the infectivity of infectious clone pSVBV-SY,and detected the viral genome DNA.The results show that the infectious clone pSVBV-SY has infectivity,and typical symptoms occur in strawberry veinbanding forest,vacuum inoculation can improve the Strawberry Virus Infectious Clone inoculation efficiency.2.Infectivity identification of SVBV virus vectorSVBV American isolate virus vector pSVBV-MCS method of vacuum filtration inoculation,55 d after forest strawberry system young leaves exhibited typical symptoms of veinbanding.PCR detection showed that pSVBV-MCS successfully infected forest strawberries.Compared with the pSVBV-US infectious clone SVBV isolated from the United States,with the inoculation of pSVBV-MCS virus vector,the infection rate was reduced,the latency time of host disease was delayed,and the phenotype of the vein was reduced.3.SVBV virus infectious clone are capable of transactivating bicistronic expressionCloning of CaMV,SVBV-US and SVBV-SY complete ORF VII gene and ORF I gene 5'terminal 51 nts fragment were inserted into vector pCAM-GFP,construct bicistronic vectors p35S-Ca71 GFP,pUSFlt-US71 GFP and pSYFlt-SY71 GFP,first cistron is the complete ORF VII gene,second cistron is ORF I gene 5' terminal 51 nts with the green fluorescent protein(GFP)fusion gene formation.SVBV American isolate infectious clone pSVBV-US and pCAM-2b were coinfiltration with bicistronic p35S-Ca71 GFP,and the results showed that the viral infectious clone could transactivate the express of the bicistronic in the Nicotiana benthamiana.4.Identification of transactivator of SVBV virusIn order to identify viral transactivation function protein expression vector was constructed containing 6 ORFs of SVBV,pCAM-USP1,pCAM-USP2,pCAM-USP3,pCAM-USP4,pCAM-USP5 and pCAM-USP6,and the bicistronic p35S-Ca71 GFP coinfiltration Nicotiana benthamiana,only pCAM-USP6 can transactivate the bicistronic expression,the rest ORF can not transactivate the bicistronic expression.The experimental results show that the SVBV P6 protein is a kind of viral transactivator,which is helpful to the translation of virus mRNA.pCAM-USP6 was coinfiltration with bicistronics p35S-Ca71 GFP,pUSFlt-US71 GFP and pSYFlt-SY71 GFP,respectively.It was found that pCAM-USP6 could transactivate the expression of the 3 kinds of bicistronics.Similarly,the same results were obtained by using SVBV Shenyang isolate pCAM-SYP6 in Nicotiana benthamiana,and pCAM-SYP6 was able to transactivate the expression of the 3 kinds of bicistronics.Furthermore,the transactivation of P6 protein was further demonstrated,and the P6 protein of different isolates of SVBV could be trans activated.5.Study on transactivation domain of P6 proteinThe core functional domain of a functional protein is important for the function of the protein itself,and the protein interacts with a variety of related pathway proteins through its core functional domain.In order to explore the transactivation domain of P6 protein,the mutant of P6 was constructed according to the three-dimensional prediction of P6 protein and the prediction of P6 nuclear localization signal.P6 mutants pCAM-P6(1-300)?pCAM-P6(301-515)? pCAM-P6(1-113)? pCAM-P6(114-429)? pCAM-P6(430-515)?pCAM-P6(?193-223)and pCAM-P6(?402-426)were coinfiltration with pUSFlt-US71 GFP,and the expression of fluorescent protein was observed after 3 days observed by laser confocal microscopy.The results showed that the transactivation of the transactivator of the P6 mutant was extremely weak and those P6 mutants could not transactivate the bicistronic expression.