| RNA of Sugarcane mosaic virus (SCMV-BJ) was extracted from purified SCMV-BJ and then applied to complementary DNA synthesis by reverse transcription. The coding region was amplified by polymerase chain reaction. The full-length cDNA clone pSCMV of SCMV-BJ was constructed from three overlapping fragments cDNA clones downstream from the bacteriophage T7 promoter. The plasmid was able to generate an in vitro transcripts corresponding to SCMV-BJ with one extra guanosine residue at the 5' terminus and a poly(A) tract at the 3' end. The uncapped and capped in vitro transcripts generated by T7 RNA polymerase were introduced into maize seedlings by mechanical inoculation. There was no symptom appeared in the inoculated plants, and we can not amplify the CP gene of SCMV-BJ by RT-PCR analysis. All the results indicated that the pSCMV was uninfectious and cannot infect maize.The HC-Pro gene of SCMV-BJ was amplified by RT-PCR, and ligated to the expression vector pET-22b(+). The recombinant plasmid pETHC was transformed into E. coli BL21(DE3) and then induced to express a fussion protein by IPTG. The results of SDS-PAGE and Western blot analysis showed that the HC gene was highly expressed by IPTG induction. The molecular weight of the recombinant protein was about 52 kD, which was immunoreactive. The antiserum with high specificity was produced when the rabbit was immunized with purified recombinant protein, and the titer of antiserum was 1:8192 estimated by antigen coating plate-ELISA (ACP-ELISA). The antiserum was useful for detection of SCMV-BJ in plant materials. The subcellular locations of SCMV-BJ HC-Pro were studied by immunogold-labeled protein A. There were immunogold labels (IGL) in the cell of sieve element and plasmodesma, indicating that the HC-Pro of SCMV-BJ was involved in cell to cell and systemic movement of SCMV-BJ.The IPTG-induced expression vector pGEXPl and pGEXP3 containing P1 gene and P3 gene of SCMV-BJ respectively were constructed by cloning SCMV-BJ P1 gene and P3 gene into the expression vector pGEX4T-3 and being transferred into E. coli BL21(DE3) pLys. The results of SDS-PAGE and Western blot showed that the specific fusion proteins whose molecular weights were about 50 kD and 64 kD. They were expressed by IPTG and were immunocompetent. The antiserums with high specificity were produced after the rabbit were immunized with purified fusion proteins, and the titers of these antiserums were both 1/8192 by ACP-ELISA.The HC-Pro, P1 and P3 gene of SCMV-BJ were ligated to the PVX based virus vector p45p46 to produce pPVXHC, pPVXPl and pPVXP3. RNA transcripts were produced by in vitro transcription from p45p46 and recombinant plasmids after linearization and mechanically inoculated onto N. benthamiana. Compared with p45p46 inoculated symptom, pPVXHC and pPVXP3 inoculation induced more severe mosaic symptom, whereas no symptom was observed on pPVXPl inoculated plant. TheHC-Pro, P1 and P3 genes were detectable in the leaves by RT-PCR. Total proteins were extracted from leaf tissues, Western blot analysis by using polyclonal antisera raised against P1 and P3 fusion proteins respectively confirmed expression of P1 and P3 proteins. Analysis of the results indicated that HC-Pro was sufficient to induce the increase in PVX pathogenicity. P1 protein did not only show the same activity, but also show the inhibitory effect. And we speculated that P3 may affect the symptom by its involvement in RNA replication and also functioned as an pathogenicity determinant. |
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