Transgenic Rice Prokaryotic Expression Of Anti-black Streaked Dwarf Virus Genome Segment S9 Of

Rice black-streaked dwarf virus (RBSDV) is the member of the genus Fijivirus, family Reoviridae. Its genome contains10double-stranded RNAs which are named S1-S10respectively according to the mobilities on PAGE gel. Genome segement S9of RBSDV consists of1900base pairs, and contains two non-overlapping open reading frames (ORFs). ORF1encodes protein P9-1, ORF2encodes protein P9-2. Rice black-streaked dwarf disease has caused the reduction of rice yield in the prevailed area, even no harvest. However, until now there has been lack of rice varieties with resistance against RBSDV.Specific primers were designed according to published sequences and were used to amplify the S9-1gene of RBSDV. The S9-1gene was inserted into pGEM-T vector and then was confirmed by sequencing. The prokaryotic expression vector pSBET-S9-1containing the S9-1gene was constructed and then was transformed into Escherichia coli BL21plysS strain. After induction with IPTG, protein p9-1was highly expressed in E.coli. The expressed protein was purified and specific antiserum against P9-1was raised in mouse. Western blot analysis showed the antisera could react strongly to extraction of RBSDV-infected rice plants but not to that of health rice plants.The full-length of RBSDV genome segment S9was cloned into the binary expression vector which was reconstructed by pBI121and pCAMBIA1301, and the plant expression vector was transformed into Agrobacterium tumefaciens EHA105. Then S9gene was transferred into Xiushui04, a Japonica rice cultivar, by Agrobacterium-mediated method. Sixty-eight transgenic plants were obtained and PCR analysis revealed that the S9gene was detected in94%of transgenic rice. Southern blotting analysis indicated that12out of20transgenic rice plants contained intact genome segement S9, and most of the transgenic plants harboured only one copy.PCR and RT-PCR analysis of T1generation plants revealed that S9gene was stable in these transgenic plants and was transcripted efficiently. Western blot analysis showed the P9-1antisera reacted to saps of T1generation plants but not to that of non-transgenic rice plant. These results indicated that the target gene was successfully translated in those generation plants.