Construction Of An Infectious Clone And Prokaryotic Expression Of Gene ORF â…¥ Of Strawberry Vein Banding Virus

Strawberry vein banding virus (SVBV) is a latent virus inducing very serious damagein strawberry plants all over the world. Recently, SVBV is detected in the Henan, Hebeiand Jilin provinces in China and causes severe losses. SVBV is classified as a member ofCaulimoviridae. SVBV has a double-stranded and circle DNA genome of approximately7.8kb and contains7ORFs which encode different functional proteins. SVBV infectiousclone was constructed to study the replication, movement of virus and the relationshipbetween virus and protein factors of host plant. In addition, pathogenicity of SVBV hasclose relationship with P6protein encoded by gene ORF â…¥ of SVBV. The gene ORF â…¥was cloned and prokaryotic expression and antiserum preparation were carried out. Thework establish the basis to research the concrete functions of P6protein and has thesignificant effect on illuminating the pathogenic mechanism of plant virus.1. Construction of an infectious clone of SVBVThe pSVBV-DE3is a clone plasmid of complete sequence of SVBV about7.8kb,containing a single BamH I and a single EcoR I. An infectious clone of SVBV wasconstructed by the two restriction enzymes.0.5SVBV about4kb was digested frompSVBV-E3via BamH I and EcoR I and was inserted into binary vector pBinPLUS viathe same enzymes, forming pBin-0.5SVBV. Then pSVBV-E3was digested by EcoR I toobtain the complete SVBV. Finally, the recombination vector pBin-1.5SVBV wasobtained by inserting complete sequence of SVBV into pBin-0.5SVBV.2. Transformation, inoculation and detection of the infectious clone of SVBVThe recombination plasmid pBin-1.5SVBV was transformed into Agrobacteriumtumefaciens GV3101and the positive clone was screened. Nicotiana benthamiana, N.tabacum var. Samsun NN, N. glutinosa and Fragaria vesca plants were inoculated by A.tumefaciens transient infiltration. Moreover, the recombination plasmid of the infectiousclone of SVBV was bombarded into indicator plants F. vesca and N. benthamiana. It wasfound that there were no symptoms appeared on four plants incubated with SVBVinfectious clone20days later. The genome DNA was extracted from the new systematicleaves of four indicator plants. Specific segments of SVBV gene could be detected in N.benthamiana, N. tabacum var. Samsun NN, N. glutinosa and F. virginiana by PCR.Wherever, the SVBV specific gene could not be detected in F. vesca and N. Benthamianabombarded with SVBV infectious clone. 3. Expression, Purification and antiserum preparation of P6protein encoded byORF â…¥ of SVBVORF â…¥ gene of SVBV was amplified by PCR and was inserted into prokaryoteexpression vector pET-SUMO. After the recombinant plasmid pET-SUMO-ORF â…¥ wastransformed into Escherichia coli BL21(DE3), the recombinant protein with aapproximate molecular weight of90kD was obtained with IPTG induction andNi2+-NTA affinity column purification. Antiserum was prepared by using the purityrecombinant protein as antigen to immunize rabbits, and its titer reached1:256000byindirect-ELISA detection. Western-blot analysis indicated that the recombinant protein ata dilution of1:64000could be detected with the prepared antiserum. Furtherindirect-ELISA testing showed that the transient expression of ORF â…¥ of Chinese isolateand American isolate of SVBV in N. benthamiana could be detected with the preparedantiserum at a higher dilution (1:512000).