Cloning, Expression And Transformation Of Barley Yellow Dwarf Virus GPV ORF5 Gene

According to the results of comparison between partial sequence of GPV and the sequences of other Luteoviruses , synthesized the oligoonucleotide which complementation the sequence of GPV ORF5 S抋s upstream primer According to the RNA sequence and encode amino acid sequence of out of ORF5 downstream , designed and synthesized annex downstream primer. Fragment of ORF5 was amplified by RT-PCR with the down-and up-stream primers. Then fragment was inserted into pGEM- T Vector, and the recombinants of pGEM- T 桹RF5 was obtained. Sequence analysis shows that GPV ORF5 contains 1326 nucleotides which can code a protein of 441 amino acids with Mr about 50 kD .Compared with other Luteoviruses, there is a highest homohlogy between GPV and RPV, the similarity is 68.1% . The nucleotide similarity of ORF5 between GPV and another BYDV such as BWLV , PLRV, PAY, SGV, GAV, MAV is 39.8%, 33.6%,30.2%~ 28.8%.~ 28.5% and 29%~According to the sequence of BYDV ORF5 gene, deduced the amino acid. Compare the amino acid of ORF5 gene between GPV and another isolates of Luteoviruses in sequence, compositions character , structuralIII)c~*~4 GPV&~ ORF5~~~ ~block . The results showed that in amino acid sequence of ORF5 there is a highest homology between GPV and RPV, the similarity is 72.4% . The amino acid similarity of ORF5 between GPV and another BYDV such as BWLV , PLRV, PAY, SGV, GAV, MAy is 40.5%, 31.4%, 25.6%~ 22.6 %.. 25.1% and 26.5%.The results also showed that in amino acid composition, character structural block, there are notability different between the GPV ORF5 andRPV ORF5.Based on the sequence of ORF5, primers for ORF5 were designed and the ORF5 gene was amplified by RT-PCR, and cloned into pGEM-3Z. By the restriction enzyme digesting /fill protrudling end! restriction enzyme digesting,the fragment having one blunt end and one protruding end of ORF5, was inserted into prokaryotic expression vector(pET-5a), the ORF5 gene was expressed without a fusion peptide with IPTG induce. The result of SDS-PAGE showed that ORF5 expressed about 5OKD protein. Western blotting indicated that the expression protein of ORF5 could be detected with antiserum of GPV.Plant expression plasmids of pPPI 15(containing ORF5) were obtained by inserting the fragments into pEmu-mcs-N plasmid. Longjian 127 and Jinmai 47 were transformed with pPPI 15 via the pollen tube pathway. The seeds harvested were detected by PCR analysis and southern blot hybridization. Three positive transgenic lines of transgene wheat generation were obtained. PCR and Southern-blotting analysis showed positive result.