CLONING AND EXPRESSION ANALYSIS OF MAJOR CAPSID PROTEIN GENE, ENDOPEPTIDASE AND HOLIN GENE OF CYANOPHAGE PAV-LD

he 142 open reading frame (ORF) of the genome of cyanophage, PaV-LD (Planktothrix agardhii virus isolated from the Lake Donghu), was elucidated recently. However, the characteristic of genomic, phylogenetic position, and mechanism of infection remained to be examined on PaV-LD, a novel tailless caynophage. With the relatively stable structure and evolutionarily conserved sequence, the major capsid protein (MCP) gene has been an important reference in the classification of these viruses. Endopeptidase and holin were considered to play an important role in the progress of invasion and release of viruses. The MCP gene (073R), endopeptidase gene (123L), and holin gene (124L) of cyanophage PaV-LD were cloned and the expressions were analysed. PaV-LD 073R was amplified and cloned, and the prokaryotic expression plasmid pET-32a-073R was constructed. The prokaryotic expression products of PaV-LD 073R was analyzed by SDS-PAGE after being induced with isopropyl beta-D-thiogalactopyranoside (IPTG). The fusion protein was purified via affinity chromatography and was then used to immunize BALB/C mice to prepare the antiserum against PaV-LD 073R. Next the antiserum was used for Western blotting analysis and the temporal expression pattern of PaV-LD 073R was characterized during PaV-LD infection at different times. The results of western blotting indicated that the MCP was initially expressed at 48h post-infection (p.i.) and developed into particularly enhanced from 60h p.i. to 84h p.i. The data also demonstrated that 073R was a late expression gene of the tailless cyanophage genome. To further investigate the phylogenetic position of PaV-LD, a phylogenetic tree was constructed using the Neighbor-Joining method with 34 tailed cyanophages and 2 phycoviruses (viruses that infect eukaryotic algae). The analysis of the phylogenetic tree revealed that the amino acid sequences in PaV-LD 073R grouped more closely with tailless phycoviruses instead of the tailed cyanophages, indicating that PaV-LD had the morphological characteristics of the tailless phycoviruses. PaV-LD endopeptidase gene and holin gene (two adjacent ORFs in the genome of PaV-LD, 123L-124L) were amplified using polymerase chain reaction (PCR) and the plasmid contained a spectinomycin resistance gene, then the promoter genes pPetE and 123L-124L were constructed and integrated into the genome of Synechocystis sp. PCC6803 via homologous recombination. The growth curves of the recombinant and the wild type of Synechocystis sp. PCC 6803 were drawn, and the growth rate of the recombinant was found to be significantly slower than the wild type. Ultrastructural morphology observation with transmission electron microscopy showed that the cell wall of recombinant was damaged partly or fully, causing protoplast swelling and the leakage of cell contents. Results suggested that PaV-LD 123L-124L could express the functional proteins that played a role in dissolving cell wall and slowing down cell growth in the recombinant Synechocystis sp. PCC 6803.