According to the genomic sequence of foreign four PLRV isolates, three pairs of specific primer were designed and synthesized. The cDNA of the ORF2a gene of PLRV-Ch was synthesized by reverse transcription and followed by Polymerase Chain Reaction amplication. The synthesized 3' and 5' cDNA fragment of the PLRV-Ch ORF2a gene were inserted into pUC19 and cloned in E. coli JM109 and were sequenced respectively. The middle cDNA fragment were directly sequenced. The homology of nucleotide sequence of PLRV-Ch compared with PLRV-S (Scotland, UK), PLRV-N(Netherlands), PLRV-A(Australia) and PLRV-C(Canada) were 98.96%, 98.70%, 94.79%, 97.5%, the homology of putative amino acid sequence are 97.97%, 97.97%, 89.69%, 95.94%. In 3' region of ORF2a gene a slippery sequence for-1 frameshift and its downstream "stem-loop" or "pseudoknot" and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with-1 frameshift. The amino acid sequence of C terminus region of 70 kD protein translated by motif IV has a protease characteristic motif and a helicase motif IV. The amino acid sequence of polypeptide translated by ORF2a gene undergoing frameshift has a single-stranded nucleic acid binding protein-like characteristic motif.
Amino Acid Motifs ; Luteovirus ; genetics ; Open Reading Frames ; genetics ; Protein Folding ; Reverse Transcriptase Polymerase Chain Reaction ; Solanum tuberosum ; virology ; Viral Proteins ; chemistry ; genetics

Abstract:By using PVX derived vector pGR107, the effect of BYDV-MP nuclear localization signal on the movement of PVX was studied. BYDV-MP was cloned into pGR107 using GFP as an indicator. BYDV-MP was then shown to induce the systemic infection and exacerbate the symptom of PVX through infecting Nicotiana benthamiana. When the PVX gene encoding 25kD protein, which functioned as a systematic movemnet protein,was deleted and the above experiment was repeated, the result showed that BYDV-MP could compensate the systemic movement of PVX. A serial mutants with substitutions on the fifth, sixth and seventh amino acids of BYDV-MP nuclear localization signal was further constructed. It was found that the mutants at the fifth, sixth amino acids in BYDV-MP nuclear localization signal could only delay or weaken systemic movement of PVX whereas the mutant at seventh amino acid could entirely inhibit systemic movement of PVX.
Amino Acid Sequence ; Green Fluorescent Proteins ; genetics ; Luteovirus ; physiology ; Molecular Sequence Data ; Nuclear Localization Signals ; chemistry ; physiology ; Plant Viral Movement Proteins ; physiology ; Potexvirus ; genetics ; physiology

According to published nucleotide sequences, ORF4 gene of barley yellow dwarf virus GAV (BYDV-GAV) was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). The BYDV-GAV ORF4 gene was expressed in baculovirus -insect cell expression system efficiently, and western bolt analysis confirmed its expression product. Confocal laser scanning microscopy showed that GFP: ORF4 fusion protein was associated with the nuclear envelope of insect cells. By expressing the N- and C-terminal regions of ORF4-encoding product (P4) in insect cells combined with structure prediction, it was found that the N-terminal region of P4 containing four a-helices is required for targeting P4 to the nuclear envelope. These results provide a base for biological function of ORF4 gene during systemic infection of BYDV-GAV in host plants further.
Amino Acid Sequence ; Animals ; Baculoviridae ; genetics ; metabolism ; Genes, Plant ; genetics ; Green Fluorescent Proteins ; genetics ; Insecta ; genetics ; metabolism ; Luteovirus ; genetics ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism